| pdb id | class | method | authors | reference | abstract | annotation |
|---|---|---|---|---|---|---|
| 10mh | transferase-DNA | X-ray (2.55 Å) | Sheikhnejad G, Brank A, Christman JK, Goddard A, Alvarez E, Ford Jr H, Marquez VE, Marasco CJ, Sufrin JR, O'gara M, Cheng X | (1999) "Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine." J.Mol.Biol., 285, 2021-2034. doi: 10.1006/jmbi.1998.2426. | A key step in the predicted mechanism of enzymatic transfer of methyl groups from S-adenosyl-l-methionine (AdoMet) to cytosine residues in DNA is the transient formation of a dihydrocytosine intermediate covalently linked to cysteine in the active site of a DNA (cytosine C5)-methyltransferase (DNA C5-MTase). Crystallographic analysis of complexes formed by HhaI methyltransferase (M.HhaI), AdoMet and a target oligodeoxyribonucleotide containing 5-fluorocytosine confirmed the existence of this dihydrocytosine intermediate. Based on the premise that 5,6-dihydro-5-azacytosine (DZCyt), a cytosine analog with an sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, could mimic the non-aromatic character of the cytosine ring in this transition state, we synthesized a series of synthetic substrates for DNA C5-MTase containing DZCyt. Substitution of DZCyt for target cytosines in C-G dinucleotides of single-stranded or double-stranded oligodeoxyribonucleotide substrates led to complete inhibition of methylation by murine DNA C5-MTase. Substitution of DZCyt for the target cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had a similar effect on methylation by M. HhaI. Oligodeoxyribonucleotides containing DZCyt formed a tight but reversible complex with M.HhaI, and were consistently more potent as inhibitors of DNA methylation than oligodeoxyribonucleotides identical in sequence containing 5-fluorocytosine. Crystallographic analysis of a ternary complex involving M.HhaI, S-adenosyl-l-homocysteine and a double-stranded 13-mer oligodeoxyribonucleotide containing DZCyt at the target position showed that the analog is flipped out of the DNA helix in the same manner as cytosine, 5-methylcytosine, and 5-fluorocytosine. However, no formation of a covalent bond was detected between the sulfur atom of the catalytic site nucleophile, cysteine 81, and the pyrimidine C6 carbon. These results indicate that DZCyt can occupy the active site of M.HhaI as a transition state mimic and, because of the high degree of affinity of its interaction with the enzyme, it can act as a potent inhibitor of methylation. | Ternary structure of hhai methyltransferase with adohcy and hemimethylated DNA containing 5,6-dihydro-5-azacytosine at the target. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 1bsu | hydrolase-DNA | X-ray (2.0 Å) | Martin AM, Sam MD, Reich NO, Perona JJ | (1999) "Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease." Nat.Struct.Biol., 6, 269-277. doi: 10.1038/6707. | Specific recognition by EcoRV endonuclease of its cognate, sharply bent GATATC site at the center TA step occurs solely via hydrophobic interaction with thymine methyl groups. Mechanistic kinetic analyses of base analog-substituted DNAs at this position reveal that direct readout provides 5 kcal mol(-1) toward specificity, with an additional 6-10 kcal mol(-1) arising from indirect readout. Crystal structures of several base analog complexes show that the major-groove hydrophobic contacts are crucial to forming required divalent metal-binding sites, and that indirect readout operates in part through the sequence-dependent free-energy cost of unstacking the center base-pair step of the DNA. | Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease. ➥[+]:Acg/cgT, [-]:cgT/Acg, is-WC-paired, is-in-duplex, other-contacts |
| 1dct | transferase-DNA | X-ray (2.8 Å) | Reinisch KM, Chen L, Verdine GL, Lipscomb WN | (1995) "The crystal structure of HaeIII methyltransferase convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing." Cell(Cambridge,Mass.), 82, 143-153. doi: 10.1016/0092-8674(95)90060-8. | Many organisms expand the information content of their genome through enzymatic methylation of cytosine residues. Here we report the 2.8 A crystal structure of a bacterial DNA (cytosine-5)-methyltransferase (DCMtase), M. HaeIII, bound covalently to DNA. In this complex, the substrate cytosine is extruded from the DNA helix and inserted into the active site of the enzyme, as has been observed for another DCMtase, M. HhaI. The DNA is bound in a cleft between the two domains of the protein and is distorted from the characteristic B-form conformation at its recognition sequence. A comparison of structures shows a variation in the mode of DNA recognition: M. HaeIII differs from M. HhaI in that the remaining bases in its recognition sequence undergo an extensive rearrangement in their pairing. In this process, the bases are unstacked, and a gap 8 A long opens in the DNA. | DNA (cytosine-5) methylase from haeiii covalently bound to DNA. ➥[-]:GGC/GcC, hydrophobic-with-AA, is-WC-paired, is-in-duplex, stacking-with-AA |
| 1ig4 | transcription-DNA | NMR | Ohki I, Shimotake N, Fujita N, Jee J, Ikegami T, Nakao M, Shirakawa M | (2001) "Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA." Cell(Cambridge,Mass.), 105, 487-497. doi: 10.1016/S0092-8674(01)00324-5. | In vertebrates, the biological consequences of DNA methylation are often mediated by protein factors containing conserved methyl-CpG binding domains (MBDs). Mutations in the MBD protein MeCP2 cause the neurodevelopmental disease Rett syndrome. We report here the solution structure of the MBD of the human methylation-dependent transcriptional regulator MBD1 bound to methylated DNA. DNA binding causes a loop in MBD1 to fold into a major and novel DNA binding interface. Recognition of the methyl groups and CG sequence at the methylation site is due to five highly conserved residues that form a hydrophobic patch. The structure indicates how MBD may access nucleosomal DNA without encountering steric interference from core histones, and provides a basis to interpret mutations linked to Rett syndrome in MeCP2. | Solution structure of the methyl-cpg-binding domain of human mbd1 in complex with methylated DNA. ➥[+]:CcG/cGG, [-]:cGG/CcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, other-contacts |
| 2c7o | transferase-DNA | X-ray (1.9 Å) | Neely RK, Daujotyte D, Grazulis S, Magennis SW, Dryden DTF, Klimasauskas S, Jones AC | (2005) "Time-Resolved Fluorescence of 2-Aminopurine as a Probe of Base Flipping in M.HhaI-DNA Complexes." Nucleic Acids Res., 33, 6953. doi: 10.1093/NAR/GKI995. | DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 A resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures. | Hhai DNA methyltransferase complex with 13mer oligonucleotide containing 2-aminopurine adjacent to the target base (pcgc:gmgc) and sah. ➥[+]:GcC/gGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 2c7p | transferase-DNA | X-ray (1.7 Å) | Neely RK, Daujotyte D, Grazulis S, Magennis SW, Dryden DTF, Klimasauskas S, Jones AC | (2005) "Time-Resolved Fluorescence of 2-Aminopurine as a Probe of Base Flipping in M.HhaI-DNA Complexes." Nucleic Acids Res., 33, 6953. doi: 10.1093/NAR/GKI995. | DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 A resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures. | Hhai DNA methyltransferase complex with oligonucleotide containing 2- aminopurine opposite to the target base (gcgc:gmpc) and sah. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 2c7q | transferase-DNA | X-ray (1.85 Å) | Neely RK, Daujotyte D, Grazulis S, Magennis SW, Dryden DTF, Klimasauskas S, Jones AC | (2005) "Time-Resolved Fluorescence of 2-Aminopurine as a Probe of Base Flipping in M.HhaI-DNA Complexes." Nucleic Acids Res., 33, 6953. doi: 10.1093/NAR/GKI995. | DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 A resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures. | Hhai DNA methyltransferase complex with oligonucleotide containing 2- aminopurine outside the recognition sequence (paired with g) and sah. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 2c7r | transferase-DNA | X-ray (1.9 Å) | Neely RK, Daujotyte D, Grazulis S, Magennis SW, Dryden DTF, Klimasauskas S, Jones AC | (2005) "Time-Resolved Fluorescence of 2-Aminopurine as a Probe of Base Flipping in M.HhaI-DNA Complexes." Nucleic Acids Res., 33, 6953. doi: 10.1093/NAR/GKI995. | DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 A resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures. | Hhai DNA methyltransferase (t250g mutant) complex with oligonucleotide containing 2-aminopurine as a target base (gpgc:gmgc) and sah. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 2ky8 | transcription-DNA | NMR | Scarsdale JN, Webb HD, Ginder GD, Williams DC | (2011) "Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence." Nucleic Acids Res., 39, 6741-6752. doi: 10.1093/nar/gkr262. | The epigenetic code of DNA methylation is interpreted chiefly by methyl cytosine binding domain (MBD) proteins which in turn recruit multiprotein co-repressor complexes. We previously isolated one such complex, MBD2-NuRD, from primary erythroid cells and have shown it contributes to embryonic/fetal β-type globin gene silencing during development. This complex has been implicated in silencing tumor suppressor genes in a variety of human tumor cell types. Here we present structural details of chicken MBD2 bound to a methylated DNA sequence from the ρ-globin promoter to which it binds in vivo and mediates developmental transcriptional silencing in normal erythroid cells. While previous studies have failed to show sequence specificity for MBD2 outside of the symmetric mCpG, we find that this domain binds in a single orientation on the ρ-globin target DNA sequence. Further, we show that the orientation and affinity depends on guanine immediately following the mCpG dinucleotide. Dynamic analyses show that DNA binding stabilizes the central β-sheet, while the N- and C-terminal regions of the protein maintain mobility. Taken together, these data lead to a model in which DNA binding stabilizes the MBD2 structure and that binding orientation and affinity is influenced by the DNA sequence surrounding the central mCpG. | Solution structure and dynamic analysis of chicken mbd2 methyl binding domain bound to a target methylated DNA sequence. ➥[+]:TcG/cGA, [-]:cGG/CcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 2moe | hydrolase-DNA | NMR | Walavalkar NM, Cramer JM, Buchwald WA, Scarsdale JN, Williams DC | (2015) "Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches." Nucleic Acids Res., 42, 11218-11232. doi: 10.1093/nar/gku782. | Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting mCpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for mCpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference for mCpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to (m)CpG islands and promotes scanning by rapidly exchanging between successive mCpG sites which facilitates repair of nearby mCpG/TpG mismatches by the glycosylase domain. | Solution structure of mbd4 methyl-cytosine binding domain bound to methylated DNA. ➥[-]:cGG/CcG, is-WC-paired, is-in-duplex, other-contacts |
| 2uyc | transferase | X-ray (2.0 Å) | Daujotyte D, Grazulis S | "HhaI DNA Methyltransferase R163N Mutant Complex with 13mer Gcgc-Gmgc Oligonucleotide and Sah." | Hhai DNA methyltransferase r163n mutant complex with 13mer gcgc-gmgc oligonucleotide and sah. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex | |
| 2uyh | transferase | X-ray (2.63 Å) | Daujotyte D, Grazulis S | "HhaI DNA Methyltransferase S87Q-Q237S Mutant Complex with 13mer Gcgc-Gmgc Oligonucleotide and Sah." | Hhai DNA methyltransferase s87q-q237s mutant complex with 13mer gcgc- gmgc oligonucleotide and sah. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex | |
| 2uz4 | transferase | X-ray (2.1 Å) | Daujotyte D, Leinartaite L, Grazulis S | "DNA Methyltransferase R165N Mutant." | Hhai DNA methyltransferase r165n mutant complex with 13mer gcgc-gmgc oligonucleotide and sah. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex | |
| 2zkd | ligase | X-ray (1.6 Å) | Arita K, Ariyoshi M, Tochio H, Nakamura Y, Shirakawa M | (2008) "Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism." Nature, 455, 818-821. doi: 10.1038/nature07249. | DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1. | Crystal structure of the sra domain of mouse np95 in complex with hemi-methylated cpg DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 2zke | ligase | X-ray (2.6 Å) | Arita K, Ariyoshi M, Tochio H, Nakamura Y, Shirakawa M | (2008) "Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism." Nature, 455, 818-821. doi: 10.1038/nature07249. | DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1. | Crystal structure of the sra domain of mouse np95 in complex with hemi-methylated cpg DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 2zkf | ligase | X-ray (2.55 Å) | Arita K, Ariyoshi M, Tochio H, Nakamura Y, Shirakawa M | (2008) "Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism." Nature, 455, 818-821. doi: 10.1038/nature07249. | DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1. | Crystal structure of the sra domain of mouse np95 in complex with hemi-methylated cpg DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 2zo0 | ligase-DNA | X-ray (2.19 Å) | Hashimoto H, Horton JR, Zhang X, Bostick M, Jacobsen SE, Cheng X | (2008) "The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix." Nature, 455, 826-829. doi: 10.1038/nature07280. | Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA. | Mouse np95 sra domain DNA specific complex 1. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 2zo1 | ligase-DNA | X-ray (1.96 Å) | Hashimoto H, Horton JR, Zhang X, Bostick M, Jacobsen SE, Cheng X | (2008) "The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix." Nature, 455, 826-829. doi: 10.1038/nature07280. | Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA. | Mouse np95 sra domain DNA specific complex 2. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3c2i | transcription regulator | X-ray (2.5 Å) | Ho KL, McNae IW, Schmiedeberg L, Klose RJ, Bird AP, Walkinshaw MD | (2008) "MeCP2 binding to DNA depends upon hydration at methyl-CpG." Mol.Cell, 29, 525-531. doi: 10.1016/j.molcel.2007.12.028. | MeCP2 is an essential transcriptional repressor that mediates gene silencing through binding to methylated DNA. Binding specificity has been thought to depend on hydrophobic interactions between cytosine methyl groups and a hydrophobic patch within the methyl-CpG-binding domain (MBD). X-ray analysis of a methylated DNA-MBD cocrystal reveals, however, that the methyl groups make contact with a predominantly hydrophilic surface that includes tightly bound water molecules. This suggests that MeCP2 recognizes hydration of the major groove of methylated DNA rather than cytosine methylation per se. The MeCP2-DNA complex also identifies a unique structural role for T158, the residue most commonly mutated in Rett syndrome. | The crystal structure of methyl-cpg binding domain of human mecp2 in complex with a methylated DNA sequence from bdnf. ➥[+]:AcG/cGT, [-]:cGG/CcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 3clz | ligase | X-ray (2.2 Å) | Avvakumov GV, Walker JR, Xue S, Li Y, Duan S, Bronner C, Arrowsmith CH, Dhe-Paganon S | (2008) "Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1." Nature, 455, 822-825. doi: 10.1038/nature07273. | Epigenetic inheritance in mammals is characterized by high-fidelity replication of CpG methylation patterns during development. UHRF1 (also known as ICBP90 in humans and Np95 in mouse) is an E3 ligase important for the maintenance of global and local DNA methylation in vivo. The preferential affinity of UHRF1 for hemi-methylated DNA over symmetrically methylated DNA by means of its SET and RING-associated (SRA) domain and its association with the maintenance DNA methyltransferase 1 (DNMT1) suggests a role in replication of the epigenetic code. Here we report the 1.7 A crystal structure of the apo SRA domain of human UHRF1 and a 2.2 A structure of its complex with hemi-methylated DNA, revealing a previously unknown reading mechanism for methylated CpG sites (mCpG). The SRA-DNA complex has several notable structural features including a binding pocket that accommodates the 5-methylcytosine that is flipped out of the duplex DNA. Two specialized loops reach through the resulting gap in the DNA from both the major and the minor grooves to read the other three bases of the CpG duplex. The major groove loop confers both specificity for the CpG dinucleotide and discrimination against methylation of deoxycytidine of the complementary strand. The structure, along with mutagenesis data, suggests how UHRF1 acts as a key factor for DNMT1 maintenance methylation through recognition of a fundamental unit of epigenetic inheritance, mCpG. | The set and ring associated (sra) domain of uhrf1 bound to methylated DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3f8i | ligase-DNA | X-ray (2.29 Å) | Hashimoto H, Horton JR, Zhang X, Cheng X | (2009) "UHRF1, a modular multi-domain protein, regulates replication-coupled crosstalk between DNA methylation and histone modifications." Epigenetics, 4, 8-14. | Cytosine methylation in DNA is a major epigenetic signal, and plays a central role in propagating chromatin status during cell division. However the mechanistic links between DNA methylation and histone methylation are poorly understood. A multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for DNA CpG maintenance methylation at replication forks, and mouse UHRF1-null cells show enhanced susceptibility to DNA replication arrest and DNA damaging agents. Recent data demonstrated that the SET and RING associated (SRA) domain of UHRF1 binds hemimethylated CpG and flips 5-methylcytosine out of the DNA helix, whereas its tandom tudor domain and PHD domain bind the tail of histone H3 in a highly methylation sensitive manner. We hypothesize that UHRF1 brings the two components (histones and DNA) carrying appropriate markers (on the tails of H3 and hemimethylated CpG sites) ready to be assembled into a nucleosome after replication. | Mouse uhrf1 sra domain bound with hemi-methylated cpg, crystal structure in space group p21. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3f8j | ligase-DNA | X-ray (1.99 Å) | Hashimoto H, Horton JR, Zhang X, Cheng X | (2009) "UHRF1, a modular multi-domain protein, regulates replication-coupled crosstalk between DNA methylation and histone modifications." Epigenetics, 4, 8-14. | Cytosine methylation in DNA is a major epigenetic signal, and plays a central role in propagating chromatin status during cell division. However the mechanistic links between DNA methylation and histone methylation are poorly understood. A multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for DNA CpG maintenance methylation at replication forks, and mouse UHRF1-null cells show enhanced susceptibility to DNA replication arrest and DNA damaging agents. Recent data demonstrated that the SET and RING associated (SRA) domain of UHRF1 binds hemimethylated CpG and flips 5-methylcytosine out of the DNA helix, whereas its tandom tudor domain and PHD domain bind the tail of histone H3 in a highly methylation sensitive manner. We hypothesize that UHRF1 brings the two components (histones and DNA) carrying appropriate markers (on the tails of H3 and hemimethylated CpG sites) ready to be assembled into a nucleosome after replication. | Mouse uhrf1 sra domain bound with hemi-methylated cpg, crystal structure in space group c222(1). ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3fde | ligase | X-ray (1.41 Å) | Hashimoto H, Horton JR, Zhang X, Cheng X | (2009) "UHRF1, a modular multi-domain protein, regulates replication-coupled crosstalk between DNA methylation and histone modifications." Epigenetics, 4, 8-14. | Cytosine methylation in DNA is a major epigenetic signal, and plays a central role in propagating chromatin status during cell division. However the mechanistic links between DNA methylation and histone methylation are poorly understood. A multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for DNA CpG maintenance methylation at replication forks, and mouse UHRF1-null cells show enhanced susceptibility to DNA replication arrest and DNA damaging agents. Recent data demonstrated that the SET and RING associated (SRA) domain of UHRF1 binds hemimethylated CpG and flips 5-methylcytosine out of the DNA helix, whereas its tandom tudor domain and PHD domain bind the tail of histone H3 in a highly methylation sensitive manner. We hypothesize that UHRF1 brings the two components (histones and DNA) carrying appropriate markers (on the tails of H3 and hemimethylated CpG sites) ready to be assembled into a nucleosome after replication. | Mouse uhrf1 sra domain bound with hemi-methylated cpg DNA, crystal structure in space group c222(1) at 1.4 Å resolution. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3q0b | transferase-DNA | X-ray (2.2 Å) | Rajakumara E, Law JA, Simanshu DK, Voigt P, Johnson LM, Reinberg D, Patel DJ, Jacobsen SE | (2011) "A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo." Genes Dev., 25, 137-152. doi: 10.1101/gad.1980311. | Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci. | Crystal structure of suvh5 sra- fully methylated cg DNA complex in space group p42212. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3q0c | transferase-DNA | X-ray (2.66 Å) | Rajakumara E, Law JA, Simanshu DK, Voigt P, Johnson LM, Reinberg D, Patel DJ, Jacobsen SE | (2011) "A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo." Genes Dev., 25, 137-152. doi: 10.1101/gad.1980311. | Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci. | Crystal structure of suvh5 sra-fully methylated cg DNA complex in space group p6122. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3q0d | transferase-DNA | X-ray (2.37 Å) | Rajakumara E, Law JA, Simanshu DK, Voigt P, Johnson LM, Reinberg D, Patel DJ, Jacobsen SE | (2011) "A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo." Genes Dev., 25, 137-152. doi: 10.1101/gad.1980311. | Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci. | Crystal structure of suvh5 sra- hemi methylated cg DNA complex. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3q0f | transferase-DNA | X-ray (2.75 Å) | Rajakumara E, Law JA, Simanshu DK, Voigt P, Johnson LM, Reinberg D, Patel DJ, Jacobsen SE | (2011) "A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo." Genes Dev., 25, 137-152. doi: 10.1101/gad.1980311. | Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci. | Crystal structure of suvh5 sra- methylated chh DNA complex. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3ssc | DNA binding protein-DNA | X-ray (2.1 Å) | Sukackaite R, Grazulis S, Tamulaitis G, Siksnys V | (2012) "The recognition domain of the methyl-specific endonuclease McrBC flips out 5-methylcytosine." Nucleic Acids Res., 40, 7552-7562. doi: 10.1093/nar/gks332. | DNA cytosine methylation is a widespread epigenetic mark. Biological effects of DNA methylation are mediated by the proteins that preferentially bind to 5-methylcytosine (5mC) in different sequence contexts. Until now two different structural mechanisms have been established for 5mC recognition in eukaryotes; however, it is still unknown how discrimination of the 5mC modification is achieved in prokaryotes. Here we report the crystal structure of the N-terminal DNA-binding domain (McrB-N) of the methyl-specific endonuclease McrBC from Escherichia coli. The McrB-N protein shows a novel DNA-binding fold adapted for 5mC-recognition. In the McrB-N structure in complex with methylated DNA, the 5mC base is flipped out from the DNA duplex and positioned within a binding pocket. Base flipping elegantly explains why McrBC system restricts only T4-even phages impaired in glycosylation [Luria, S.E. and Human, M.L. (1952) A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol., 64, 557-569]: flipped out 5-hydroxymethylcytosine is accommodated in the binding pocket but there is no room for the glycosylated base. The mechanism for 5mC recognition employed by McrB-N is highly reminiscent of that for eukaryotic SRA domains, despite the differences in their protein folds. | DNA binding domain of restriction endonuclease bound to DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3ssd | DNA binding protein-DNA | X-ray (2.2 Å) | Sukackaite R, Grazulis S, Tamulaitis G, Siksnys V | (2012) "The recognition domain of the methyl-specific endonuclease McrBC flips out 5-methylcytosine." Nucleic Acids Res., 40, 7552-7562. doi: 10.1093/nar/gks332. | DNA cytosine methylation is a widespread epigenetic mark. Biological effects of DNA methylation are mediated by the proteins that preferentially bind to 5-methylcytosine (5mC) in different sequence contexts. Until now two different structural mechanisms have been established for 5mC recognition in eukaryotes; however, it is still unknown how discrimination of the 5mC modification is achieved in prokaryotes. Here we report the crystal structure of the N-terminal DNA-binding domain (McrB-N) of the methyl-specific endonuclease McrBC from Escherichia coli. The McrB-N protein shows a novel DNA-binding fold adapted for 5mC-recognition. In the McrB-N structure in complex with methylated DNA, the 5mC base is flipped out from the DNA duplex and positioned within a binding pocket. Base flipping elegantly explains why McrBC system restricts only T4-even phages impaired in glycosylation [Luria, S.E. and Human, M.L. (1952) A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol., 64, 557-569]: flipped out 5-hydroxymethylcytosine is accommodated in the binding pocket but there is no room for the glycosylated base. The mechanism for 5mC recognition employed by McrB-N is highly reminiscent of that for eukaryotic SRA domains, despite the differences in their protein folds. | DNA binding domain of restriction endonuclease bound to DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 3vxv | hydrolase-DNA | X-ray (2.0 Å) | Otani J, Arita K, Kato T, Kinoshita M, Kimura H, Suetake I, Tajima S, Ariyoshi M, Shirakawa M | (2013) "Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4." J.Biol.Chem., 288, 6351-6362. doi: 10.1074/jbc.M112.431098. | The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides ((5m)CG/(5m)CG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine ((5m)CG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with (5m)CG/(5m)CG and (5m)CG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine. | Crystal structure of methyl cpg binding domain of mbd4 in complex with the 5mcg-tg sequence. ➥[+]:CcG/TGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 3vxx | hydrolase-DNA | X-ray (2.204 Å) | Otani J, Arita K, Kato T, Kinoshita M, Kimura H, Suetake I, Tajima S, Ariyoshi M, Shirakawa M | (2013) "Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4." J.Biol.Chem., 288, 6351-6362. doi: 10.1074/jbc.M112.431098. | The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides ((5m)CG/(5m)CG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine ((5m)CG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with (5m)CG/(5m)CG and (5m)CG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine. | Crystal structure of methyl cpg binding domain of mbd4 in complex with the 5mcg-5mcg sequence. ➥[+]:CcG/cGG, [-]:cGG/CcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 3vyb | hydrolase-DNA | X-ray (2.4 Å) | Otani J, Arita K, Kato T, Kinoshita M, Kimura H, Suetake I, Tajima S, Ariyoshi M, Shirakawa M | (2013) "Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4." J.Biol.Chem., 288, 6351-6362. doi: 10.1074/jbc.M112.431098. | The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides ((5m)CG/(5m)CG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine ((5m)CG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with (5m)CG/(5m)CG and (5m)CG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine. | Crystal structure of methyl cpg binding domain of mbd4 in complex with the 5mcg-hmcg sequence. ➥[+]:CcG/cGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 3vyq | hydrolase-DNA | X-ray (2.525 Å) | Otani J, Arita K, Kato T, Kinoshita M, Kimura H, Suetake I, Tajima S, Ariyoshi M, Shirakawa M | (2013) "Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4." J.Biol.Chem., 288, 6351-6362. doi: 10.1074/jbc.M112.431098. | The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides ((5m)CG/(5m)CG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine ((5m)CG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with (5m)CG/(5m)CG and (5m)CG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine. | Crystal structure of the methyl cpg binding domain of mbd4 in complex with the 5mcg-tg sequence in space group p1. ➥[+]:CcG/TGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 4aqu | hydrolase | X-ray (2.3 Å) | Valton J, Daboussi F, Leduc S, Molina R, Redondo P, Macmaster R, Montoya G, Duchateau P | (2012) "5'-Cytosine-Phosphoguanine (Cpg) Methylation Impacts the Activity of Natural and Engineered Meganucleases." J.Biol.Chem., 287, 30139. doi: 10.1074/JBC.M112.379966. | In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions. | Crystal structure of i-crei complexed with its target methylated at position plus 2 (in the b strand) in the presence of calcium. ➥[-]:CGT/AcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 4aqx | hydrolase | X-ray (2.2 Å) | Valton J, Daboussi F, Leduc S, Molina R, Redondo P, Macmaster R, Montoya G, Duchateau P | (2012) "5'-Cytosine-Phosphoguanine (Cpg) Methylation Impacts the Activity of Natural and Engineered Meganucleases." J.Biol.Chem., 287, 30139. doi: 10.1074/JBC.M112.379966. | In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions. | Crystal structure of i-crei complexed with its target methylated at position plus 2 (in the b strand) in the presence of magnesium. ➥[-]:CGT/AcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 4da4 | transferase-DNA | X-ray (2.6 Å) | Song J, Teplova M, Ishibe-Murakami S, Patel DJ | (2012) "Structure-Based Mechanistic Insights into DNMT1-Mediated Maintenance DNA Methylation." Science, 335, 709-712. doi: 10.1126/science.1214453. | DNMT1, the major maintenance DNA methyltransferase in animals, helps to regulate gene expression, genome imprinting, and X-chromosome inactivation. We report on the crystal structure of a productive covalent mouse DNMT1(731-1602)-DNA complex containing a central hemimethylated CpG site. The methyl group of methylcytosine is positioned within a shallow hydrophobic concave surface, whereas the cytosine on the target strand is looped out and covalently anchored within the catalytic pocket. The DNA is distorted at the hemimethylated CpG step, with side chains from catalytic and recognition loops inserting through both grooves to fill an intercalation-type cavity associated with a dual base flip-out on partner strands. Structural and biochemical data establish how a combination of active and autoinhibitory mechanisms ensures the high fidelity of DNMT1-mediated maintenance DNA methylation. | Structure of mouse dnmt1 (731-1602) bound to hemimethylated cpg DNA. ➥[+]:CcG/GGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 4dkj | transferase-DNA | X-ray (2.15 Å) | Wojciechowski M, Czapinska H, Bochtler M | (2013) "CpG underrepresentation and the bacterial CpG-specific DNA methyltransferase M.MpeI." Proc.Natl.Acad.Sci.USA, 110, 105-110. doi: 10.1073/pnas.1207986110. | Cytosine methylation promotes deamination. In eukaryotes, CpG methylation is thought to account for CpG underrepresentation. Whether scarcity of CpGs in prokaryotic genomes is diagnostic for methylation is not clear. Here, we report that Mycoplasms tend to be CpG depleted and to harbor a family of constitutively expressed or phase variable CpG-specific DNA methyltransferases. The very CpG poor Mycoplasma penetrans and its constitutively active CpG-specific methyltransferase M.MpeI were chosen for further characterization. Genome-wide sequencing of bisulfite-converted DNA indicated that M.MpeI methylated CpG target sites both in vivo and in vitro in a locus-nonselective manner. A crystal structure of M.MpeI with DNA at 2.15-Å resolution showed that the substrate base was flipped and that its place in the DNA stack was taken by a glutamine residue. A phenylalanine residue was intercalated into the "weak" CpG step of the nonsubstrate strand, indicating mechanistic similarities in the recognition of the short CpG target sequence by prokaryotic and eukaryotic DNA methyltransferases. | Cpg specific methyltransferase in complex with target DNA. ➥[-]:GGC/GcC, is-WC-paired, is-in-duplex, stacking-with-AA |
| 4f6n | DNA binding protein-DNA | X-ray (2.8 Å) | Buck-Koehntop BA, Stanfield RL, Ekiert DC, Martinez-Yamout MA, Dyson HJ, Wilson IA, Wright PE | (2012) "Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso." Proc.Natl.Acad.Sci.USA, 109, 15229-15234. doi: 10.1073/pnas.1213726109. | Methylation of CpG dinucleotides in DNA is a common epigenetic modification in eukaryotes that plays a central role in maintenance of genome stability, gene silencing, genomic imprinting, development, and disease. Kaiso, a bifunctional Cys(2)His(2) zinc finger protein implicated in tumor-cell proliferation, binds to both methylated CpG (mCpG) sites and a specific nonmethylated DNA motif (TCCTGCNA) and represses transcription by recruiting chromatin remodeling corepression machinery to target genes. Here we report structures of the Kaiso zinc finger DNA-binding domain in complex with its nonmethylated, sequence-specific DNA target (KBS) and with a symmetrically methylated DNA sequence derived from the promoter region of E-cadherin. Recognition of specific bases in the major groove of the core KBS and mCpG sites is accomplished through both classical and methyl CH···O hydrogen-bonding interactions with residues in the first two zinc fingers, whereas residues in the C-terminal extension following the third zinc finger bind in the opposing minor groove and are required for high-affinity binding. The C-terminal region is disordered in the free protein and adopts an ordered structure upon binding to DNA. The structures of these Kaiso complexes provide insights into the mechanism by which a zinc finger protein can recognize mCpG sites as well as a specific, nonmethylated regulatory DNA sequence. | Crystal structure of kaiso zinc finger DNA binding protein in complex with methylated cpg site DNA. ➥[+]:CcG/cGG, [+]:GcG/cGc, [-]:cGT/AcG, [-]:cGc/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 4gjp | transcription-DNA | X-ray (1.94 Å) | Yan N, Deng D, Yan CY, Yin P, Pan XJ, Shi YG | "Crystal structure of a protein complex." | Crystal structure of the tal effector dhax3 bound to dsDNA containing repetitive methyl-cpg. ➥[+]:GcG/CGC, [+]:TcG/CGA, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 4gjr | transcription-DNA | X-ray (1.85 Å) | Yan N, Deng D, Yan CY, Yin P, Pan XJ, Shi YG | "Crystal structure of a protein complex." | Crystal structure of the tal effector dhax3 bound to methylated dsDNA. ➥[+]:AcC/GGT, [+]:CcC/GGG, [+]:TcT/AGA, hydrophobic-with-AA, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 4gzn | transcription-DNA | X-ray (0.99 Å) | Liu Y, Toh H, Sasaki H, Zhang X, Cheng X | (2012) "An atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence." Genes Dev., 26, 2374-2379. doi: 10.1101/gad.202200.112. | Zinc finger transcription factor Zfp57 recognizes the methylated CpG within the TGCCGC element. We determined the structure of the DNA-binding domain of Zfp57, consisting of two adjacent zinc fingers, in complex with fully methylated DNA at 1.0 Å resolution. The first zinc finger contacts the 5' half (TGC), and the second recognizes the 3' half (CGC) of the recognition sequence. Zfp57 recognizes the two 5-methylcytosines (5mCs) asymmetrically: One involves hydrophobic interactions with Arg178, which also interacts with the neighboring 3' guanine and forms a 5mC-Arg-G interaction, while the other involves a layer of ordered water molecules. Two point mutations in patients with transient neonatal diabetes abolish DNA-binding activity. Zfp57 has reduced binding affinity for unmodified DNA and the oxidative products of 5mC. | Mouse zfp57 zinc fingers in complex with methylated DNA. ➥[+]:CcG/cGG, [-]:cGC/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 4hp1 | DNA binding protein-DNA | X-ray (2.25 Å) | Xu Y, Xu C, Kato A, Tempel W, Abreu JG, Bian C, Hu Y, Hu D, Zhao B, Cerovina T, Diao J, Wu F, He HH, Cui Q, Clark E, Ma C, Barbara A, Veenstra GJ, Xu G, Kaiser UB, Liu XS, Sugrue SP, He X, Min J, Kato Y, Shi YG | (2012) "Tet3 CXXC Domain and Dioxygenase Activity Cooperatively Regulate Key Genes for Xenopus Eye and Neural Development." Cell(Cambridge,Mass.), 151, 1200-1213. doi: 10.1016/j.cell.2012.11.014. | Ten-Eleven Translocation (Tet) family of dioxygenases dynamically regulates DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functions and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further demonstrate that the Tet3 CXXC domain is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are both crucial for Tet3's biological function. Together, these findings define Tet3 as a transcription regulator and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development. | Crystal structure of tet3 in complex with a non-cpg dsDNA. ➥[+]:CcG/cGG, [-]:cGG/CcG, is-WC-paired, is-in-duplex, other-contacts |
| 4lg7 | hydrolase-DNA | X-ray (2.5 Å) | Xu C, Tempel W, Wernimont AK, Bountra C, Arrowsmith CH, Edwards AM, Min J | "Crystal structure MBD4 MBD domain in complex with methylated CpG DNA." | Crystal structure mbd4 mbd domain in complex with methylated cpg DNA. ➥[+]:AcG/cGT, [-]:cGT/AcG, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 4lt5 | oxidoreductase-DNA | X-ray (2.893 Å) | Hashimoto H, Pais JE, Zhang X, Saleh L, Fu ZQ, Dai N, Correa IR, Zheng Y, Cheng X | (2014) "Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA." Nature, 506, 391-395. doi: 10.1038/nature12905. | Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(ii)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla. | Structure of a naegleria tet-like dioxygenase in complex with 5-methylcytosine DNA. ➥[+]:GcT/AGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex, not-WC-paired, not-in-duplex, stacking-with-AA |
| 4m9e | transcription-DNA | X-ray (1.851 Å) | Liu Y, Olanrewaju YO, Zheng Y, Hashimoto H, Blumenthal RM, Zhang X, Cheng X | (2014) "Structural basis for Klf4 recognition of methylated DNA." Nucleic Acids Res., 42, 4859-4867. doi: 10.1093/nar/gku134. | Transcription factor Krüppel-like factor 4 (Klf4), one of the factors directing cellular reprogramming, recognizes the CpG dinucleotide (whether methylated or unmodified) within a specific G/C-rich sequence. The binding affinity of the mouse Klf4 DNA-binding domain for methylated DNA is only slightly stronger than that for an unmodified oligonucleotide. The structure of the C-terminal three Krüppel-like zinc fingers (ZnFs) of mouse Klf4, in complex with fully methylated DNA, was determined at 1.85 Å resolution. An arginine and a glutamate interact with the methyl group. By comparison with two other recently characterized structures of ZnF protein complexes with methylated DNA, we propose a common principle of recognition of methylated CpG by C2H2 ZnF proteins, which involves a spatially conserved Arg-Glu pair. | Structure of klf4 zinc finger DNA binding domain in complex with methylated DNA. ➥[+]:GcG/cGC, [-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 4m9v | transcription-DNA | X-ray (0.969 Å) | Liu Y, Olanrewaju YO, Zhang X, Cheng X | (2013) "DNA recognition of 5-carboxylcytosine by a zfp57 mutant at an atomic resolution of 0.97 angstrom." Biochemistry, 52, 9310-9317. doi: 10.1021/bi401360n. | The Zfp57 gene encodes a KRAB (Krüppel-associated box) domain-containing C2H2 zinc finger transcription factor that is expressed in early development. Zfp57 protein recognizes methylated CpG dinucleotide within GCGGCA elements at multiple imprinting control regions. In the previously determined structure of the mouse Zfp57 DNA-binding domain in complex with DNA containing 5-methylcytosine (5mC), the side chains of Arg178 and Glu182 contact the methyl group via hydrophobic and van der Waals interactions. We examined the role of Glu182 in recognition of 5mC by mutagenesis. The majority of mutants examined lose selectivity of methylated (5mC) over unmodified (C) and oxidative derivatives, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC), suggesting that the side chain of Glu182 (the size and the charge) is dispensable for methyl group recognition but negatively impacts the binding of unmodified cytosine as well as oxidized derivatives of 5mC to achieve 5mC selectivity. Substitution of Glu182 with its corresponding amide (E182Q) had no effect on methylated DNA binding but gained significant binding affinity for 5caC DNA, resulting in a binding affinity for 5caC DNA comparable to that of the wild-type protein for 5mC. We show structurally that the uncharged amide group of E182Q interacts favorably with the carboxylate group of 5caC. Furthermore, introducing a positively charged arginine at position 182 resulted in a mutant (E182R) having higher selectivity for the negatively charged 5caC. | Zfp57 mutant (e182q) in complex with 5-carboxylcytosine DNA. ➥[+]:CcG/cGG, is-WC-paired, is-in-duplex, other-contacts |
| 4mht | transferase-DNA | X-ray (2.7 Å) | O'Gara M, Klimasauskas S, Roberts RJ, Cheng X | (1996) "Enzymatic C5-cytosine methylation of DNA: mechanistic implications of new crystal structures for HhaL methyltransferase-DNA-AdoHcy complexes." J.Mol.Biol., 261, 634-645. doi: 10.1006/jmbi.1996.0489. | The refined crystal structures of HhaI methyltransferase complexed with cognate unmethylated or methylated DNA together with S-adenosyl-L-homocysteine, along with the previously-solved binary and covalent ternary structures, offer a detailed picture of the active site at individual stages throughout the reaction cycle. This picture supports and extends a proposed mechanism for C5-cytosine methylation that may be general for the whole family of C5-cytosine methyltransferases. The structures of the two new complexes have been refined to crystallographic R-factors of 0.189 and 0.178, respectively, at 2.7 A resolution. We observe that both unmethylated 2'-deoxycytidine and 5-methyl-2'-deoxycytidine flip out of the DNA helix and fit into the active site of the enzyme. The catalytic sulfur atom of Cys81 interacts strongly with C6. The C5 methyl group of the flipped 5-methyl-2'-deoxycytidine is bent approximately 50 degrees out of the plane of the cytosine ring and towards the sulfur atom of S-adenosyl-L-homocysteine. This unusual position is probably due to partial sp3 character at C5 and C6 and to steric effects of the conserved amino acid residues Pro80 and Cys81. Two water molecules are held near the hydrophobic edge (C5 and C6) of the flipped cytosine by two conserved amino acid residues (Gln82 and Asn304) and the phosphoryl oxygen atom of the phosphate group 3' to the flipped nucleotide, and one of them may serve as the general base for eliminating the proton from C5. Protonation of the cytosine N3 during the methylation reaction may involve Glu119, which itself might be protonated via a water-mediated interaction between the terminal carboxyl group of Glu119 and the amino group of the methionine moiety of S-adenosyl-L-methionine. The cofactor thus plays two key roles in the reaction. | Ternary structure of hhai methyltransferase with native DNA and adohcy. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex, not-WC-paired, not-in-duplex, stacking-with-AA |
| 4nm6 | oxidoreductase-DNA | X-ray (2.026 Å) | Hu L, Li Z, Cheng J, Rao Q, Gong W, Liu M, Shi YG, Zhu J, Wang P, Xu Y | (2013) "Crystal Structure of TET2-DNA Complex: Insight into TET-Mediated 5mC Oxidation." Cell(Cambridge,Mass.), 155, 1545-1555. doi: 10.1016/j.cell.2013.11.020. | TET proteins oxidize 5-methylcytosine (5mC) on DNA and play important roles in various biological processes. Mutations of TET2 are frequently observed in myeloid malignance. Here, we present the crystal structure of human TET2 bound to methylated DNA at 2.02 Å resolution. The structure shows that two zinc fingers bring the Cys-rich and DSBH domains together to form a compact catalytic domain. The Cys-rich domain stabilizes the DNA above the DSBH core. TET2 specifically recognizes CpG dinucleotide and shows substrate preference for 5mC in a CpG context. 5mC is inserted into the catalytic cavity with the methyl group orientated to catalytic Fe(II) for reaction. The methyl group is not involved in TET2-DNA contacts so that the catalytic cavity allows TET2 to accommodate 5mC derivatives for further oxidation. Mutations of Fe(II)/NOG-chelating, DNA-interacting, and zinc-chelating residues are frequently observed in human cancers. Our studies provide a structural basis for understanding the mechanisms of TET-mediated 5mC oxidation. | Crystal structure of tet2-DNA complex. ➥[-]:.GG/Cc., is-WC-paired, is-in-duplex, not-WC-paired, not-in-duplex, stacking-with-AA |
| 4pw7 | ligase-DNA | X-ray (2.001 Å) | Zhou T, Xiong J, Wang M, Yang N, Wong J, Zhu B, Xu RM | (2014) "Structural Basis for Hydroxymethylcytosine Recognition by the SRA Domain of UHRF2." Mol.Cell, 54, 879-886. doi: 10.1016/j.molcel.2014.04.003. | Methylated cytosine of CpG dinucleotides in vertebrates may be oxidized by Tet proteins, a process that can lead to DNA demethylation. The predominant oxidation product, 5-hydroxymethylcytosine (5hmC), has been implicated in embryogenesis, cell differentiation, and human diseases. Recently, the SRA domain of UHRF2 (UHRF2-SRA) has been reported to specifically recognize 5hmC, but how UHRF2 recognizes this modification is unclear. Here we report the structure of UHRF2-SRA in complex with a 5hmC-containing DNA. The structure reveals that the conformation of a phenylalanine allows the formation of an optimal 5hmC binding pocket, and a hydrogen bond between the hydroxyl group of 5hmC and UHRF2-SRA is critical for their preferential binding. Further structural and biochemical analyses unveiled the role of SRA domains as a versatile reader of modified DNA, and the knowledge should facilitate further understanding of the biological function of UHRF2 and the comprehension of DNA hydroxymethylation in general. | Structure of uhrf2-sra in complex with a 5mc-containing DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 4qen | transcription-DNA | X-ray (2.002 Å) | Du J, Johnson LM, Groth M, Feng S, Hale CJ, Li S, Vashisht AA, Gallego-Bartolome J, Wohlschlegel JA, Patel DJ, Jacobsen SE | (2014) "Mechanism of DNA Methylation-Directed Histone Methylation by KRYPTONITE." Mol.Cell, 55, 495-504. doi: 10.1016/j.molcel.2014.06.009. | In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide, and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the ε-ammonium of K9 positioned adjacent to bound SAH. These structural insights, complemented by functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation. | Crystal structure of kryptonite in complex with mchh DNA and sah. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 4qeo | transcription-DNA | X-ray (2.0 Å) | Du J, Johnson LM, Groth M, Feng S, Hale CJ, Li S, Vashisht AA, Gallego-Bartolome J, Wohlschlegel JA, Patel DJ, Jacobsen SE | (2014) "Mechanism of DNA Methylation-Directed Histone Methylation by KRYPTONITE." Mol.Cell, 55, 495-504. doi: 10.1016/j.molcel.2014.06.009. | In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide, and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the ε-ammonium of K9 positioned adjacent to bound SAH. These structural insights, complemented by functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation. | Crystal structure of kryptonite in complex with mchh DNA, h3(1-15) peptide and sah. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 4qep | transcription-DNA | X-ray (3.1 Å) | Du J, Johnson LM, Groth M, Feng S, Hale CJ, Li S, Vashisht AA, Gallego-Bartolome J, Wohlschlegel JA, Patel DJ, Jacobsen SE | (2014) "Mechanism of DNA Methylation-Directed Histone Methylation by KRYPTONITE." Mol.Cell, 55, 495-504. doi: 10.1016/j.molcel.2014.06.009. | In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide, and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the ε-ammonium of K9 positioned adjacent to bound SAH. These structural insights, complemented by functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation. | Crystal structure of kryptonite in complex with mchg DNA and sah. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 4r28 | hydrolase-DNA | X-ray (3.055 Å) | Horton JR, Wang H, Mabuchi MY, Zhang X, Roberts RJ, Zheng Y, Wilson GG, Cheng X | (2014) "Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix." Nucleic Acids Res., 42, 12092-12101. doi: 10.1093/nar/gku871. | MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act. | Mspji restriction endonuclease in complex with 27-mer oligonucleotide. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 4r2a | DNA binding protein-DNA | X-ray (1.591 Å) | Hashimoto H, Olanrewaju YO, Zheng Y, Wilson GG, Zhang X, Cheng X | (2014) "Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence." Genes Dev., 28, 2304-2313. doi: 10.1101/gad.250746.114. | In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC is a major epigenetic signal that acts to regulate gene expression. 5hmC, 5fC, and 5caC are oxidized derivatives that might also act as distinct epigenetic signals. We investigated the response of the zinc finger DNA-binding domains of transcription factors early growth response protein 1 (Egr1) and Wilms tumor protein 1 (WT1) to different forms of modified cytosine within their recognition sequence, 5'-GCG(T/G)GGGCG-3'. Both displayed high affinity for the sequence when C or 5mC was present and much reduced affinity when 5hmC or 5fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5caC affected the two proteins differently, abolishing binding by Egr1 but not by WT1. We ascribe this difference to electrostatic interactions in the binding sites. In Egr1, a negatively charged glutamate conflicts with the negatively charged carboxylate of 5caC, whereas the corresponding glutamine of WT1 interacts with this group favorably. Our analyses shows that zinc finger proteins (and their splice variants) can respond in modulated ways to alternative modifications within their binding sequence. | Egr1-zif268 zinc fingers in complex with methylated DNA. ➥[+]:GcG/cGC, [-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 4r2e | DNA binding protein-DNA | X-ray (1.84 Å) | Hashimoto H, Olanrewaju YO, Zheng Y, Wilson GG, Zhang X, Cheng X | (2014) "Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence." Genes Dev., 28, 2304-2313. doi: 10.1101/gad.250746.114. | In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC is a major epigenetic signal that acts to regulate gene expression. 5hmC, 5fC, and 5caC are oxidized derivatives that might also act as distinct epigenetic signals. We investigated the response of the zinc finger DNA-binding domains of transcription factors early growth response protein 1 (Egr1) and Wilms tumor protein 1 (WT1) to different forms of modified cytosine within their recognition sequence, 5'-GCG(T/G)GGGCG-3'. Both displayed high affinity for the sequence when C or 5mC was present and much reduced affinity when 5hmC or 5fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5caC affected the two proteins differently, abolishing binding by Egr1 but not by WT1. We ascribe this difference to electrostatic interactions in the binding sites. In Egr1, a negatively charged glutamate conflicts with the negatively charged carboxylate of 5caC, whereas the corresponding glutamine of WT1 interacts with this group favorably. Our analyses shows that zinc finger proteins (and their splice variants) can respond in modulated ways to alternative modifications within their binding sequence. | Wilms tumor protein (wt1) zinc fingers in complex with methylated DNA. ➥[+]:GcG/cGC, [-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 4r2r | DNA binding protein-DNA | X-ray (2.089 Å) | Hashimoto H, Olanrewaju YO, Zheng Y, Wilson GG, Zhang X, Cheng X | (2014) "Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence." Genes Dev., 28, 2304-2313. doi: 10.1101/gad.250746.114. | In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC is a major epigenetic signal that acts to regulate gene expression. 5hmC, 5fC, and 5caC are oxidized derivatives that might also act as distinct epigenetic signals. We investigated the response of the zinc finger DNA-binding domains of transcription factors early growth response protein 1 (Egr1) and Wilms tumor protein 1 (WT1) to different forms of modified cytosine within their recognition sequence, 5'-GCG(T/G)GGGCG-3'. Both displayed high affinity for the sequence when C or 5mC was present and much reduced affinity when 5hmC or 5fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5caC affected the two proteins differently, abolishing binding by Egr1 but not by WT1. We ascribe this difference to electrostatic interactions in the binding sites. In Egr1, a negatively charged glutamate conflicts with the negatively charged carboxylate of 5caC, whereas the corresponding glutamine of WT1 interacts with this group favorably. Our analyses shows that zinc finger proteins (and their splice variants) can respond in modulated ways to alternative modifications within their binding sequence. | Wilms tumor protein (wt1) zinc fingers in complex with carboxylated DNA. ➥[-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts |
| 4r2s | DNA binding protein-DNA | X-ray (2.489 Å) | Hashimoto H, Olanrewaju YO, Zheng Y, Wilson GG, Zhang X, Cheng X | (2014) "Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence." Genes Dev., 28, 2304-2313. doi: 10.1101/gad.250746.114. | In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC is a major epigenetic signal that acts to regulate gene expression. 5hmC, 5fC, and 5caC are oxidized derivatives that might also act as distinct epigenetic signals. We investigated the response of the zinc finger DNA-binding domains of transcription factors early growth response protein 1 (Egr1) and Wilms tumor protein 1 (WT1) to different forms of modified cytosine within their recognition sequence, 5'-GCG(T/G)GGGCG-3'. Both displayed high affinity for the sequence when C or 5mC was present and much reduced affinity when 5hmC or 5fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5caC affected the two proteins differently, abolishing binding by Egr1 but not by WT1. We ascribe this difference to electrostatic interactions in the binding sites. In Egr1, a negatively charged glutamate conflicts with the negatively charged carboxylate of 5caC, whereas the corresponding glutamine of WT1 interacts with this group favorably. Our analyses shows that zinc finger proteins (and their splice variants) can respond in modulated ways to alternative modifications within their binding sequence. | Wilms tumor protein (wt1) q369p zinc fingers in complex with methylated DNA. ➥[+]:GcG/cGC, [-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 4x9j | transcription regulator-DNA | X-ray (1.412 Å) | Zandarashvili L, White MA, Esadze A, Iwahara J | (2015) "Structural impact of complete CpG methylation within target DNA on specific complex formation of the inducible transcription factor Egr-1." Febs Lett., 589, 1748-1753. doi: 10.1016/j.febslet.2015.05.022. | The inducible transcription factor Egr-1 binds specifically to 9-bp target sequences containing two CpG sites that can potentially be methylated at four cytosine bases. Although it appears that complete CpG methylation would make an unfavorable steric clash in the previous crystal structures of the complexes with unmethylated or partially methylated DNA, our affinity data suggest that DNA recognition by Egr-1 is insensitive to CpG methylation. We have determined, at a 1.4-Å resolution, the crystal structure of the Egr-1 zinc-finger complex with completely methylated target DNA. Structural comparison of the three different methylation states reveals why Egr-1 can recognize the target sequences regardless of CpG methylation. | Egr-1 with doubly methylated DNA. ➥[+]:GcG/cGC, [-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 5bt2 | DNA binding protein-DNA | X-ray (2.2 Å) | Chia JY, Tan WS, Ng CL, Hu NJ, Foo HL, Ho KL | (2016) "A/T Run Geometry of B-form DNA Is Independent of Bound Methyl-CpG Binding Domain, Cytosine Methylation and Flanking Sequence." Sci Rep, 6, 31210. doi: 10.1038/srep31210. | DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed. | Mecp2 mbd domain (a140v) in complex with methylated DNA. ➥[+]:AcG/cGT, [-]:cGG/CcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 5cg9 | oxidoreductase-DNA | X-ray (2.693 Å) | Hashimoto H, Pais JE, Dai N, Correa IR, Zhang X, Zheng Y, Cheng X | (2015) "Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA." Nucleic Acids Res., 43, 10713-10721. doi: 10.1093/nar/gkv870. | The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solved NgTet1-5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably because the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate. | Ngtet1 in complex with 5mc DNA in space group p3221. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 5cpj | "structural protein-DNA | X-ray (3.15 Å) | Osakabe A, Adachi F, Arimura Y, Maehara K, Ohkawa Y, Kurumizaka H | (2015) "Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA." Open Biology, 5. doi: 10.1098/rsob.150128. | DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells. | Nucleosome containing methylated sat2r DNA. ➥[+]:TcG/cGA, [-]:cGA/TcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, other-contacts |
| 5cpk | structural protein-DNA | X-ray (2.632 Å) | Osakabe A, Adachi F, Arimura Y, Maehara K, Ohkawa Y, Kurumizaka H | (2015) "Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA." Open Biology, 5. doi: 10.1098/rsob.150128. | DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells. | Nucleosome containing methylated sat2l DNA. ➥[+]:TcG/cGA, is-WC-paired, is-in-duplex, other-contacts |
| 5ef6 | transcription | X-ray (3.0 Å) | Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, Das PK, Kivioja T, Dave K, Zhong F, Nitta KR, Taipale M, Popov A, Ginno PA, Domcke S, Yan J, Schubeler D, Vinson C, Taipale J | (2017) "Impact of cytosine methylation on DNA binding specificities of human transcription factors." Science, 356. doi: 10.1126/science.aaj2239. | The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences. | Structure of hoxb13 complex with methylated DNA. ➥[+]:AcG/cGT, [-]:cGA/TcG, [-]:cGG/CcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 5ego | transcription | X-ray (2.54 Å) | Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, Das PK, Kivioja T, Dave K, Zhong F, Nitta KR, Taipale M, Popov A, Ginno PA, Domcke S, Yan J, Schubeler D, Vinson C, Taipale J | (2017) "Impact of cytosine methylation on DNA binding specificities of human transcription factors." Science, 356. doi: 10.1126/science.aaj2239. | The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences. | Hoxb13-meis1 heterodimer bound to methylated DNA. ➥[+]:AcG/cGT, [-]:cGA/TcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 5emc | transcription-DNA | X-ray (2.3 Å) | Jin J, Lian T, SU XD | "The effects of cytosine methylation on general transcription factors." | Transcription factor grdbd and smgre complex. ➥[-]:TGT/AcA, is-WC-paired, is-in-duplex, other-contacts | |
| 5gse | structural protein-DNA | X-ray (3.14 Å) | Kato D, Osakabe A, Arimura Y, Mizukami Y, Horikoshi N, Saikusa K, Akashi S, Nishimura Y, Park SY, Nogami J, Maehara K, Ohkawa Y, Matsumoto A, Kono H, Inoue R, Sugiyama M, Kurumizaka H | (2017) "Crystal structure of the overlapping dinucleosome composed of hexasome and octasome." Science, 356, 205-208. doi: 10.1126/science.aak9867. | Nucleosomes are dynamic entities that are repositioned along DNA by chromatin remodeling processes. A nucleosome repositioned by the switch-sucrose nonfermentable (SWI/SNF) remodeler collides with a neighbor and forms the intermediate "overlapping dinucleosome." Here, we report the crystal structure of the overlapping dinucleosome, in which two nucleosomes are associated, at 3.14-angstrom resolution. In the overlapping dinucleosome structure, the unusual "hexasome" nucleosome, composed of the histone hexamer lacking one H2A-H2B dimer from the conventional histone octamer, contacts the canonical "octasome" nucleosome, and they intimately associate. Consequently, about 250 base pairs of DNA are left-handedly wrapped in three turns, without a linker DNA segment between the hexasome and octasome moieties. The overlapping dinucleosome structure may provide important information to understand how nucleosome repositioning occurs during the chromatin remodeling process. | Crystal structure of unusual nucleosome. ➥[+]:CcT/AGG, is-WC-paired, is-in-duplex, other-contacts |
| 5ke7 | transcription-DNA | X-ray (2.06 Å) | Hashimoto H, Wang D, Steves AN, Jin P, Blumenthal RM, Zhang X, Cheng X | (2016) "Distinctive Klf4 mutants determine preference for DNA methylation status." Nucleic Acids Res., 44, 10177-10185. doi: 10.1093/nar/gkw774. | Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay. | Mouse klf4 znf1-3 and tpg-mpa sequence DNA complex structure. ➥[-]:TGT/AcA, is-WC-paired, is-in-duplex, other-contacts |
| 5ke8 | transcription-DNA | X-ray (2.45 Å) | Hashimoto H, Wang D, Steves AN, Jin P, Blumenthal RM, Zhang X, Cheng X | (2016) "Distinctive Klf4 mutants determine preference for DNA methylation status." Nucleic Acids Res., 44, 10177-10185. doi: 10.1093/nar/gkw774. | Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay. | Mouse klf4 e446p znf1-3 and mpg-mpg sequence DNA complex structure. ➥[+]:GcG/cGC, [-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 5kl4 | transcription-DNA | X-ray (1.783 Å) | Hashimoto H, Zhang X, Zheng Y, Wilson GG, Cheng X | (2016) "Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications." Nucleic Acids Res., 44, 10165-10176. doi: 10.1093/nar/gkw766. | Mutations in human zinc-finger transcription factor WT1 result in abnormal development of the kidneys and genitalia and an array of pediatric problems including nephropathy, blastoma, gonadal dysgenesis and genital discordance. Several overlapping phenotypes are associated with WT1 mutations, including Wilms tumors, Denys-Drash syndrome (DDS), Frasier syndrome (FS) and WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). These conditions vary in severity from individual to individual; they can be fatal in early childhood, or relatively benign into adulthood. DDS mutations cluster predominantly in zinc fingers (ZF) 2 and 3 at the C-terminus of WT1, which together with ZF4 determine the sequence-specificity of DNA binding. We examined three DDS associated mutations in ZF2 of human WT1 where the normal glutamine at position 369 is replaced by arginine (Q369R), lysine (Q369K) or histidine (Q369H). These mutations alter the sequence-specificity of ZF2, we find, changing its affinity for certain bases and certain epigenetic forms of cytosine. X-ray crystallography of the DNA binding domains of normal WT1, Q369R and Q369H in complex with preferred sequences revealed the molecular interactions responsible for these affinity changes. DDS is inherited in an autosomal dominant fashion, implying a gain of function by mutant WT1 proteins. This gain, we speculate, might derive from the ability of the mutant proteins to sequester WT1 into unproductive oligomers, or to erroneously bind to variant target sequences. | Wilms tumor protein (wt1) znf2-4 q369h in complex with formylated DNA. ➥[-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts |
| 5kl5 | transcription-DNA | X-ray (2.289 Å) | Hashimoto H, Zhang X, Zheng Y, Wilson GG, Cheng X | (2016) "Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications." Nucleic Acids Res., 44, 10165-10176. doi: 10.1093/nar/gkw766. | Mutations in human zinc-finger transcription factor WT1 result in abnormal development of the kidneys and genitalia and an array of pediatric problems including nephropathy, blastoma, gonadal dysgenesis and genital discordance. Several overlapping phenotypes are associated with WT1 mutations, including Wilms tumors, Denys-Drash syndrome (DDS), Frasier syndrome (FS) and WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). These conditions vary in severity from individual to individual; they can be fatal in early childhood, or relatively benign into adulthood. DDS mutations cluster predominantly in zinc fingers (ZF) 2 and 3 at the C-terminus of WT1, which together with ZF4 determine the sequence-specificity of DNA binding. We examined three DDS associated mutations in ZF2 of human WT1 where the normal glutamine at position 369 is replaced by arginine (Q369R), lysine (Q369K) or histidine (Q369H). These mutations alter the sequence-specificity of ZF2, we find, changing its affinity for certain bases and certain epigenetic forms of cytosine. X-ray crystallography of the DNA binding domains of normal WT1, Q369R and Q369H in complex with preferred sequences revealed the molecular interactions responsible for these affinity changes. DDS is inherited in an autosomal dominant fashion, implying a gain of function by mutant WT1 proteins. This gain, we speculate, might derive from the ability of the mutant proteins to sequester WT1 into unproductive oligomers, or to erroneously bind to variant target sequences. | Wilms tumor protein (wt1) znf2-4 q369h in complex with carboxylated DNA. ➥[-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts |
| 5kl7 | transcription-DNA | X-ray (1.579 Å) | Hashimoto H, Zhang X, Zheng Y, Wilson GG, Cheng X | (2016) "Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications." Nucleic Acids Res., 44, 10165-10176. doi: 10.1093/nar/gkw766. | Mutations in human zinc-finger transcription factor WT1 result in abnormal development of the kidneys and genitalia and an array of pediatric problems including nephropathy, blastoma, gonadal dysgenesis and genital discordance. Several overlapping phenotypes are associated with WT1 mutations, including Wilms tumors, Denys-Drash syndrome (DDS), Frasier syndrome (FS) and WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). These conditions vary in severity from individual to individual; they can be fatal in early childhood, or relatively benign into adulthood. DDS mutations cluster predominantly in zinc fingers (ZF) 2 and 3 at the C-terminus of WT1, which together with ZF4 determine the sequence-specificity of DNA binding. We examined three DDS associated mutations in ZF2 of human WT1 where the normal glutamine at position 369 is replaced by arginine (Q369R), lysine (Q369K) or histidine (Q369H). These mutations alter the sequence-specificity of ZF2, we find, changing its affinity for certain bases and certain epigenetic forms of cytosine. X-ray crystallography of the DNA binding domains of normal WT1, Q369R and Q369H in complex with preferred sequences revealed the molecular interactions responsible for these affinity changes. DDS is inherited in an autosomal dominant fashion, implying a gain of function by mutant WT1 proteins. This gain, we speculate, might derive from the ability of the mutant proteins to sequester WT1 into unproductive oligomers, or to erroneously bind to variant target sequences. | Wilms tumor protein (wt1) znf2-4q369r in complex with carboxylated DNA. ➥[-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts |
| 5lty | transcription | X-ray (2.66 Å) | Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, Das PK, Kivioja T, Dave K, Zhong F, Nitta KR, Taipale M, Popov A, Ginno PA, Domcke S, Yan J, Schubeler D, Vinson C, Taipale J | (2017) "Impact of cytosine methylation on DNA binding specificities of human transcription factors." Science, 356. doi: 10.1126/science.aaj2239. | The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences. | Homeobox transcription factor cdx2 bound to methylated DNA. ➥[+]:AcG/cGT, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 5lux | transcription | X-ray (3.23 Å) | Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, Das PK, Kivioja T, Dave K, Zhong F, Nitta KR, Taipale M, Popov A, Ginno PA, Domcke S, Yan J, Schubeler D, Vinson C, Taipale J | (2017) "Impact of cytosine methylation on DNA binding specificities of human transcription factors." Science, 356. doi: 10.1126/science.aaj2239. | The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences. | Homeobox transcription factor cdx1 bound to methylated DNA. ➥[+]:AcG/cGT, [-]:cGA/TcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 5mcv | transcription | X-ray (1.6 Å) | Golovenko D, Brauning B, Vyas P, Haran TE, Rozenberg H, Shakked Z | (2018) "New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins." Structure, 26, 1237-1250.e6. doi: 10.1016/j.str.2018.06.006. | The tumor suppressor p53 acts as a transcription factor recognizing diverse DNA response elements (REs). Previous structural studies of p53-DNA complexes revealed non-canonical Hoogsteen geometry of A/T base pairs at conserved CATG motifs leading to changes in DNA shape and its interface with p53. To study the effects of DNA shape on binding characteristics, we designed REs with modified base pairs "locked" into either Hoogsteen or Watson-Crick form. Here we present crystal structures of these complexes and their thermodynamic and kinetic parameters, demonstrating that complexes with Hoogsteen base pairs are stabilized relative to those with all-Watson-Crick base pairs. CATG motifs are abundant in p53REs such as GADD45 and p53R2 related to cell-cycle arrest and DNA repair. The high-resolution structures of these complexes validate their propensity to adopt the unique Hoogsteen-induced structure, thus providing insights into the functional role of DNA shape and broadening the mechanisms that contribute to DNA recognition by proteins. | New insights into the role of DNA shape on its recognition by p53 proteins (complex p53dbd-lwc1). ➥[+]:gcG/Cgc, [-]:Cgc/gcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 5mcw | transcription | X-ray (1.897 Å) | Golovenko D, Brauning B, Vyas P, Haran TE, Rozenberg H, Shakked Z | (2018) "New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins." Structure, 26, 1237-1250.e6. doi: 10.1016/j.str.2018.06.006. | The tumor suppressor p53 acts as a transcription factor recognizing diverse DNA response elements (REs). Previous structural studies of p53-DNA complexes revealed non-canonical Hoogsteen geometry of A/T base pairs at conserved CATG motifs leading to changes in DNA shape and its interface with p53. To study the effects of DNA shape on binding characteristics, we designed REs with modified base pairs "locked" into either Hoogsteen or Watson-Crick form. Here we present crystal structures of these complexes and their thermodynamic and kinetic parameters, demonstrating that complexes with Hoogsteen base pairs are stabilized relative to those with all-Watson-Crick base pairs. CATG motifs are abundant in p53REs such as GADD45 and p53R2 related to cell-cycle arrest and DNA repair. The high-resolution structures of these complexes validate their propensity to adopt the unique Hoogsteen-induced structure, thus providing insights into the functional role of DNA shape and broadening the mechanisms that contribute to DNA recognition by proteins. | New insights into the role of DNA shape on its recognition by p53 proteins (complex p53dbd-lwc2). ➥[+]:gcG/Cgc, [-]:Cgc/gcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 5mht | transferase-DNA | X-ray (2.7 Å) | O'Gara M, Roberts RJ, Cheng X | (1996) "A structural basis for the preferential binding of hemimethylated DNA by HhaI DNA methyltransferase." J.Mol.Biol., 263, 597-606. doi: 10.1006/jmbi.1996.0601. | The crystal structure of HhaI methyltransferase complexed with non-palindromic duplex DNA, containing a hemimethylated recognition sequence, and with the cofactor analog S-adenosyl-L-homocysteine (AdoHcy), has been determined. The structure provides an explanation for the stronger affinities of DNA methyltransferases for hemimethylated DNA than for unmethylated or fully methylated DNA in the presence of AdoHcy. The unmethylated target 2'-deoxycytidine flips out of the DNA helix and the CH group at position 5 makes van der Waals' contacts with the sulfur atom of AdoHcy. Selectivity/preference for hemimethylated over fully methylated DNA may thus reflect interactions among the chemical substituent (H or CH3) at the C5 position of the flipped cytosine, protein and the bound AdoHcy. The 5-methyl-2'-deoxycytidine on the complementary strand remains in the DNA helix, with the methyl group almost perpendicular to the carboxylate group of Glu239, which is part of the sequence recognition loop. Thus, selectivity/preference for hemimethylated over unmethylated DNA appears to result largely from van der Waals' contacts between the planar Glu239 carboxylate and the methyl group of the 5-methyl-2'-deoxycytidine. Furthermore, the positive electrostatic potential originating from the bound AdoHcy extends to the DNA phosphate groups flanking the flipped cytosine. The increased binding to DNA by long-range electrostatic interactions should also occur with the methyl donor S-adenosyl-L-methionine. | Ternary structure of hhai methyltransferase with hemimethylated DNA and adohcy. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 5szx | transcription regulator-DNA | X-ray (2.251 Å) | Hong S, Wang D, Horton JR, Zhang X, Speck SH, Blumenthal RM, Cheng X | (2017) "Methyl-dependent and spatial-specific DNA recognition by the orthologous transcription factors human AP-1 and Epstein-Barr virus Zta." Nucleic Acids Res., 45, 2503-2515. doi: 10.1093/nar/gkx057. | Epstein-barr virus zta DNA binding domain homodimer in complex with methylated DNA. ➥[+]:TcG/cGA, [-]:cGC/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA | |
| 5t00 | transcription regulator-DNA | X-ray (2.19 Å) | Hashimoto H, Wang D, Horton JR, Zhang X, Corces VG, Cheng X | (2017) "Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA." Mol. Cell, 66, 711-720.e3. doi: 10.1016/j.molcel.2017.05.004. | The multidomain CCCTC-binding factor (CTCF), containing a tandem array of 11 zinc fingers (ZFs), modulates the three-dimensional organization of chromatin. We crystallized the human CTCF DNA-binding domain in complex with a known CTCF-binding site. While ZF2 does not make sequence-specific contacts, each finger of ZF3-7 contacts three bases of the 15-bp consensus sequence. Each conserved nucleotide makes base-specific hydrogen bonds with a particular residue. Most of the variable base pairs within the core sequence also engage in interactions with the protein. These interactions compensate for deviations from the consensus sequence, allowing CTCF to adapt to sequence variations. CTCF is sensitive to cytosine methylation at position 2, but insensitive at position 12 of the 15-bp core sequence. These differences can be rationalized structurally. Although included in crystallizations, ZF10 and ZF11 are not visible, while ZF8 and ZF9 span the backbone of the DNA duplex, conferring no sequence specificity but adding to overall binding stability. | Human ctcf znf3-7 and methylated DNA complex. ➥[+]:GcG/cGC, [-]:cGC/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 5t01 | transcription regulator-DNA | X-ray (1.89 Å) | Hong S, Wang D, Horton JR, Zhang X, Speck SH, Blumenthal RM, Cheng X | (2017) "Methyl-dependent and spatial-specific DNA recognition by the orthologous transcription factors human AP-1 and Epstein-Barr virus Zta." Nucleic Acids Res., 45, 2503-2515. doi: 10.1093/nar/gkx057. | Human c-jun DNA binding domain homodimer in complex with methylated DNA. ➥[-]:CGT/AcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex | |
| 5vmu | transcription-DNA | X-ray (2.346 Å) | Nikolova EN, Stanfield RL, Dyson HJ, Wright PE | (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. doi: 10.1021/acs.biochem.8b00065. | Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression. | Kaiso (zbtb33) zinc finger DNA binding domain in complex with a double cpg-methylated DNA resembling the specific kaiso binding sequence (kbs). ➥[+]:CcG/cGG, [+]:GcG/cGc, [-]:cGA/TcG, [-]:cGc/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 5vmv | transcription-DNA | X-ray (2.313 Å) | Nikolova EN, Stanfield RL, Dyson HJ, Wright PE | (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. doi: 10.1021/acs.biochem.8b00065. | Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression. | Kaiso (zbtb33) zinc finger DNA binding domain in complex with its double cpg-methylated DNA consensus binding site. ➥[+]:GcG/cGc, [+]:TcG/cGA, [-]:cGA/TcG, [-]:cGc/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 5vmw | transcription-DNA | X-ray (2.397 Å) | Nikolova EN, Stanfield RL, Dyson HJ, Wright PE | (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. doi: 10.1021/acs.biochem.8b00065. | Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression. | Kaiso (zbtb33) zinc finger DNA binding domain in complex with a double cpg-methylated DNA resembling the specific kaiso binding sequence (kbs). ➥[+]:CcG/cGG, [+]:GcG/cGc, [-]:cGA/TcG, [-]:cGc/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 5vmx | transcription-DNA | X-ray (2.05 Å) | Nikolova EN, Stanfield RL, Dyson HJ, Wright PE | (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. doi: 10.1021/acs.biochem.8b00065. | Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression. | Kaiso (zbtb33) zinc finger DNA binding domain in complex with a hemi cpg-methylated DNA resembling the specific kaiso binding sequence (kbs). ➥[+]:CcG/CGG, [+]:GcG/CGC, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 5vmy | transcription-DNA | X-ray (2.002 Å) | Nikolova EN, Stanfield RL, Dyson HJ, Wright PE | (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. doi: 10.1021/acs.biochem.8b00065. | Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression. | Kaiso (zbtb33) zinc finger DNA binding domain in complex with a hemi cpg-methylated DNA resembling the specific kaiso binding sequence (kbs). ➥[+]:CcG/CGG, [+]:GcG/CGC, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 5vmz | transcription-DNA | X-ray (2.319 Å) | Nikolova EN, Stanfield RL, Dyson HJ, Wright PE | (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. doi: 10.1021/acs.biochem.8b00065. | Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression. | Kaiso (zbtb33) e535q mutant zinc finger DNA binding domain in complex with a double cpg-methylated DNA resembling the specific kaiso binding sequence (kbs). ➥[+]:CcG/cGG, [+]:GcG/cGc, [-]:cGA/TcG, [-]:cGc/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 6a5n | gene regulation-DNA | X-ray (2.4 Å) | Li X, Harris CJ, Zhong Z, Chen W, Liu R, Jia B, Wang Z, Li S, Jacobsen SE, Du J | (2018) "Mechanistic insights into plant SUVH family H3K9 methyltransferases and their binding to context-biased non-CG DNA methylation." Proc. Natl. Acad. Sci. U.S.A., 115, E8793-E8802. doi: 10.1073/pnas.1809841115. | DNA methylation functions in gene silencing and the maintenance of genome integrity. In plants, non-CG DNA methylation is linked through a self-reinforcing loop with histone 3 lysine 9 dimethylation (H3K9me2). The plant-specific SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG (SUVH) family H3K9 methyltransferases (MTases) bind to DNA methylation marks and catalyze H3K9 methylation. Here, we analyzed the structure and function of Arabidopsis thaliana SUVH6 to understand how this class of enzyme maintains methylation patterns in the genome. We reveal that SUVH6 has a distinct 5-methyl-dC (5mC) base-flipping mechanism involving a thumb loop element. Autoinhibition of H3 substrate entry is regulated by a SET domain loop, and a conformational transition in the post-SET domain upon cofactor binding may control catalysis. In vitro DNA binding and in vivo ChIP-seq data reveal that the different SUVH family H3K9 MTases have distinct DNA binding preferences, targeting H3K9 methylation to sites with different methylated DNA sequences, explaining the context biased non-CG DNA methylation in plants. | Crystal structure of arabidopsis thaliana suvh6 in complex with methylated DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 6c1a | DNA binding protein-DNA | X-ray (2.05 Å) | Liu K, Xu C, Lei M, Yang A, Loppnau P, Hughes TR, Min J | (2018) "Structural basis for the ability of MBD domains to bind methyl-CG and TG sites in DNA." J. Biol. Chem., 293, 7344-7354. doi: 10.1074/jbc.RA118.001785. | Cytosine methylation is a well characterized epigenetic mark and occurs at both CG and non-CG sites in DNA. Both methylated CG (mCG)- and mCH (H = A, C, or T)-containing DNAs, especially mCAC-containing DNAs, are recognized by methyl-CpG-binding protein 2 (MeCP2) to regulate gene expression in neuron development. However, the molecular mechanism involved in the binding of methyl-CpG-binding domain (MBD) of MeCP2 to these different DNA motifs is unclear. Here, we systematically characterized the DNA-binding selectivity of the MBDs in MeCP2 and MBD1-4 with isothermal titration calorimetry-based binding assays, mutagenesis studies, and X-ray crystallography. We found that the MBD domains of MeCP2 and MBD1-4 bind mCG-containing DNAs independently of the sequence outside the mCG dinucleotide. Moreover, some 1 MBDs bound to both methylated and unmethylated CA dinucleotide- containing DNAs, with a preference for the CAC sequence motif. We also found that MBD domains bind to mCA or nonmethylated CA DNA by recognizing the complementary TG dinucleotide, which is consistent with an overlooked ligand of MeCP2, i.e., the matrix/scaffold attachment regions (MARs/SARs) with a consensus sequence of 5'-GGTGT-3', which was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCAand hmCA-containing dsDNAs. In summary, our results suggest that in addition to mCG sites, unmethylated CA or TG sites also serve as DNA-binding sites for MeCP2 and other MBDcontaining proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. |
Mbd2 in complex with methylated DNA. ➥[-]:TGT/AcA, is-WC-paired, is-in-duplex, other-contacts |
| 6c1t | DNA binding protein-DNA | X-ray (1.84 Å) | Liu K, Xu C, Lei M, Yang A, Loppnau P, Hughes TR, Min J | (2018) "Structural basis for the ability of MBD domains to bind methyl-CG and TG sites in DNA." J. Biol. Chem., 293, 7344-7354. doi: 10.1074/jbc.RA118.001785. | Cytosine methylation is a well characterized epigenetic mark and occurs at both CG and non-CG sites in DNA. Both methylated CG (mCG)- and mCH (H = A, C, or T)-containing DNAs, especially mCAC-containing DNAs, are recognized by methyl-CpG-binding protein 2 (MeCP2) to regulate gene expression in neuron development. However, the molecular mechanism involved in the binding of methyl-CpG-binding domain (MBD) of MeCP2 to these different DNA motifs is unclear. Here, we systematically characterized the DNA-binding selectivity of the MBDs in MeCP2 and MBD1-4 with isothermal titration calorimetry-based binding assays, mutagenesis studies, and X-ray crystallography. We found that the MBD domains of MeCP2 and MBD1-4 bind mCG-containing DNAs independently of the sequence outside the mCG dinucleotide. Moreover, some 1 MBDs bound to both methylated and unmethylated CA dinucleotide- containing DNAs, with a preference for the CAC sequence motif. We also found that MBD domains bind to mCA or nonmethylated CA DNA by recognizing the complementary TG dinucleotide, which is consistent with an overlooked ligand of MeCP2, i.e., the matrix/scaffold attachment regions (MARs/SARs) with a consensus sequence of 5'-GGTGT-3', which was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCAand hmCA-containing dsDNAs. In summary, our results suggest that in addition to mCG sites, unmethylated CA or TG sites also serve as DNA-binding sites for MeCP2 and other MBDcontaining proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. |
Mbd2 in complex with a partially methylated DNA. ➥[+]:AcA/TGT, is-WC-paired, is-in-duplex, other-contacts |
| 6c1u | DNA binding protein-DNA | X-ray (2.3 Å) | Liu K, Xu C, Lei M, Yang A, Loppnau P, Hughes TR, Min J | (2018) "Structural basis for the ability of MBD domains to bind methyl-CG and TG sites in DNA." J. Biol. Chem., 293, 7344-7354. doi: 10.1074/jbc.RA118.001785. | Cytosine methylation is a well characterized epigenetic mark and occurs at both CG and non-CG sites in DNA. Both methylated CG (mCG)- and mCH (H = A, C, or T)-containing DNAs, especially mCAC-containing DNAs, are recognized by methyl-CpG-binding protein 2 (MeCP2) to regulate gene expression in neuron development. However, the molecular mechanism involved in the binding of methyl-CpG-binding domain (MBD) of MeCP2 to these different DNA motifs is unclear. Here, we systematically characterized the DNA-binding selectivity of the MBDs in MeCP2 and MBD1-4 with isothermal titration calorimetry-based binding assays, mutagenesis studies, and X-ray crystallography. We found that the MBD domains of MeCP2 and MBD1-4 bind mCG-containing DNAs independently of the sequence outside the mCG dinucleotide. Moreover, some 1 MBDs bound to both methylated and unmethylated CA dinucleotide- containing DNAs, with a preference for the CAC sequence motif. We also found that MBD domains bind to mCA or nonmethylated CA DNA by recognizing the complementary TG dinucleotide, which is consistent with an overlooked ligand of MeCP2, i.e., the matrix/scaffold attachment regions (MARs/SARs) with a consensus sequence of 5'-GGTGT-3', which was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCAand hmCA-containing dsDNAs. In summary, our results suggest that in addition to mCG sites, unmethylated CA or TG sites also serve as DNA-binding sites for MeCP2 and other MBDcontaining proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. |
Mbd2 in complex with a deoxy-oligonucleotide. ➥[-]:TGT/AcA, is-WC-paired, is-in-duplex, other-contacts |
| 6c1y | DNA binding protein-DNA | X-ray (2.3 Å) | Lei M, Tempel W, Chen S, Liu K, Min J | (2019) "Plasticity at the DNA recognition site of the MeCP2 mCG-binding domain." Biochim Biophys Acta Gene Regul Mech, 1862, 194409. doi: 10.1016/j.bbagrm.2019.194409. | MeCP2 is an abundant protein, involved in transcriptional repression by binding to CG and non-CG methylated DNA. However, MeCP2 might also function as a transcription activator as MeCP2 is found bound to sparsely methylated promoters of actively expressed genes. Furthermore, Attachment Region Binding Protein (ARBP), the chicken ortholog of MeCP2, has been reported to bind to Matrix/scaffold attachment regions (MARs/SARs) DNA with an unmethylated 5'-CAC/GTG-3' consensus sequence. In our previous study, although we have systemically measured the binding abilities of MBDs to unmethylated CAC/GTG DNA and the complex structures reveal that the MBD2-MBD (MBD of MBD2) binds to the unmethylated CAC/GTG DNA by recognizing the complementary GTG trinucleotide, how the MeCP2-MBD (MBD of MeCP2) recognizes the unmethylated CAC/GTG DNA, especially the MARs DNA, is still unclear. In this study, we investigated the binding characteristics of MeCP2 in recognizing unmethylated 5'-CAC/GTG-3' motif containing DNA by binding and structural studies. We found that MeCP2-MBD binds to MARs DNA with a comparable binding affinity to mCG DNA, and the MeCP2-CAC/GTG complex structure revealed that MeCP2 residues R111 and R133 form base-specific interactions with the GTG motif. For comparison, we also determined crystal structures of the MeCP2-MBD bound to mCG and mCAC/GTG DNA, respectively. Together, these crystal structures illustrate the adaptability of the MeCP2-MBD toward the GTG motif as well as the mCG DNA, and also provide structural basis of a biological role of MeCP2 as a transcription activator and its disease implications in Rett syndrome. | Mbd of human mecp2 in complex with methylated DNA. ➥[+]:CcG/cGG, [-]:cGG/CcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6c2f | DNA binding protein-DNA | X-ray (2.65 Å) | Liu K, Xu C, Tempel W, Arrowsmith CH, Bountra C, Edwards AM, Min J, Structural Genomics Consortium | "MBD2 in complex with methylated DNA." | Mbd2 in complex with methylated DNA. ➥[+]:CcG/CGG, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 6cc8 | DNA binding protein-DNA | X-ray (1.95 Å) | Liu K, Lei M, Wu Z, Gan B, Cheng H, Li Y, Min J | (2019) "Structural analyses reveal that MBD3 is a methylated CG binder." Febs J., 286, 3240-3254. doi: 10.1111/febs.14850. | The MBD3, a methyl-CpG-binding domain (MBD)-containing protein, is a core subunit of the Mi-2/NuRD complex. Recent reports show that MBD3 recognizes both methylated CG (mCG)- and hydroxymethylated CG (hmCG)-containing DNA, with a preference for hmCG. However, whether the MBD3-MBD indeed has methyl-CG-binding ability is controversial. In this study, we provided the structural basis to support the ability of MBD3-MBD to bind mCG-containing DNA. We found that the MBD3-MBD bound to mCG-containing DNA through two conserved arginine fingers, and preferentially bound to mCG over hmCG, similar to other methyl-CpG-binding MBD proteins. Compared to its closest homolog MBD2, the tyrosine-to-phenylalanine substitution at Phe34 of MBD3 is responsible for a weaker mCG DNA binding ability. Based on the complex structure of MBD3-MBD with a nonpalindromic AmCGC DNA, we suggest that all the mCG-binding MBD domains can recognize mCG-containing DNA without orientation selectivity, consistent with our observations that the sequences outside the mCG dinucleotide do not affect mCG DNA binding significantly. DNA cytosine methylation is evolutionarily conserved in most metazoans, and most invertebrates have only one MBD gene, MBD2/3. We also looked into the mCG DNA binding ability of some invertebrates MBD2/3 and found that the conserved arginine fingers and a conserved structural fold are required for methylated DNA binding by MBD2/3-MBDs in invertebrates. Hence, our results demonstrate that mCG-binding arginine fingers embedded into a conserved structural fold are essential structural features for MBD2/3s binding to methylated DNA among metazoans. | Crystal structure mbd3 mbd domain in complex with methylated cpg DNA. ➥[+]:AcG/cGT, [-]:cGT/AcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6ccg | transcription | X-ray (1.9 Å) | Liu K, Lei M, Wu Z, Gan B, Cheng H, Li Y, Min J | (2019) "Structural analyses reveal that MBD3 is a methylated CG binder." Febs J., 286, 3240-3254. doi: 10.1111/febs.14850. | The MBD3, a methyl-CpG-binding domain (MBD)-containing protein, is a core subunit of the Mi-2/NuRD complex. Recent reports show that MBD3 recognizes both methylated CG (mCG)- and hydroxymethylated CG (hmCG)-containing DNA, with a preference for hmCG. However, whether the MBD3-MBD indeed has methyl-CG-binding ability is controversial. In this study, we provided the structural basis to support the ability of MBD3-MBD to bind mCG-containing DNA. We found that the MBD3-MBD bound to mCG-containing DNA through two conserved arginine fingers, and preferentially bound to mCG over hmCG, similar to other methyl-CpG-binding MBD proteins. Compared to its closest homolog MBD2, the tyrosine-to-phenylalanine substitution at Phe34 of MBD3 is responsible for a weaker mCG DNA binding ability. Based on the complex structure of MBD3-MBD with a nonpalindromic AmCGC DNA, we suggest that all the mCG-binding MBD domains can recognize mCG-containing DNA without orientation selectivity, consistent with our observations that the sequences outside the mCG dinucleotide do not affect mCG DNA binding significantly. DNA cytosine methylation is evolutionarily conserved in most metazoans, and most invertebrates have only one MBD gene, MBD2/3. We also looked into the mCG DNA binding ability of some invertebrates MBD2/3 and found that the conserved arginine fingers and a conserved structural fold are required for methylated DNA binding by MBD2/3-MBDs in invertebrates. Hence, our results demonstrate that mCG-binding arginine fingers embedded into a conserved structural fold are essential structural features for MBD2/3s binding to methylated DNA among metazoans. | Crystal structure mbd3 mbd domain in complex with methylated cpg DNA. ➥[+]:GcG/cGC, [-]:cGC/GcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6ceu | transcription-DNA | X-ray (2.005 Å) | Liu K, Lei M, Wu Z, Gan B, Cheng H, Li Y, Min J | (2019) "Structural analyses reveal that MBD3 is a methylated CG binder." Febs J., 286, 3240-3254. doi: 10.1111/febs.14850. | The MBD3, a methyl-CpG-binding domain (MBD)-containing protein, is a core subunit of the Mi-2/NuRD complex. Recent reports show that MBD3 recognizes both methylated CG (mCG)- and hydroxymethylated CG (hmCG)-containing DNA, with a preference for hmCG. However, whether the MBD3-MBD indeed has methyl-CG-binding ability is controversial. In this study, we provided the structural basis to support the ability of MBD3-MBD to bind mCG-containing DNA. We found that the MBD3-MBD bound to mCG-containing DNA through two conserved arginine fingers, and preferentially bound to mCG over hmCG, similar to other methyl-CpG-binding MBD proteins. Compared to its closest homolog MBD2, the tyrosine-to-phenylalanine substitution at Phe34 of MBD3 is responsible for a weaker mCG DNA binding ability. Based on the complex structure of MBD3-MBD with a nonpalindromic AmCGC DNA, we suggest that all the mCG-binding MBD domains can recognize mCG-containing DNA without orientation selectivity, consistent with our observations that the sequences outside the mCG dinucleotide do not affect mCG DNA binding significantly. DNA cytosine methylation is evolutionarily conserved in most metazoans, and most invertebrates have only one MBD gene, MBD2/3. We also looked into the mCG DNA binding ability of some invertebrates MBD2/3 and found that the conserved arginine fingers and a conserved structural fold are required for methylated DNA binding by MBD2/3-MBDs in invertebrates. Hence, our results demonstrate that mCG-binding arginine fingers embedded into a conserved structural fold are essential structural features for MBD2/3s binding to methylated DNA among metazoans. | Mbd3 mbd in complex with methylated, non-palindromic cpg DNA: alternative interpretation of crystallographic data. ➥[+]:GcG/cGC, [-]:cGT/AcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6cnp | transcription-DNA | X-ray (2.1 Å) | Liu K, Xu C, Lei M, Yang A, Loppnau P, Hughes TR, Min J | (2018) "Structural basis for the ability of MBD domains to bind methyl-CG and TG sites in DNA." J. Biol. Chem., 293, 7344-7354. doi: 10.1074/jbc.RA118.001785. | Cytosine methylation is a well characterized epigenetic mark and occurs at both CG and non-CG sites in DNA. Both methylated CG (mCG)- and mCH (H = A, C, or T)-containing DNAs, especially mCAC-containing DNAs, are recognized by methyl-CpG-binding protein 2 (MeCP2) to regulate gene expression in neuron development. However, the molecular mechanism involved in the binding of methyl-CpG-binding domain (MBD) of MeCP2 to these different DNA motifs is unclear. Here, we systematically characterized the DNA-binding selectivity of the MBDs in MeCP2 and MBD1-4 with isothermal titration calorimetry-based binding assays, mutagenesis studies, and X-ray crystallography. We found that the MBD domains of MeCP2 and MBD1-4 bind mCG-containing DNAs independently of the sequence outside the mCG dinucleotide. Moreover, some 1 MBDs bound to both methylated and unmethylated CA dinucleotide- containing DNAs, with a preference for the CAC sequence motif. We also found that MBD domains bind to mCA or nonmethylated CA DNA by recognizing the complementary TG dinucleotide, which is consistent with an overlooked ligand of MeCP2, i.e., the matrix/scaffold attachment regions (MARs/SARs) with a consensus sequence of 5'-GGTGT-3', which was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCAand hmCA-containing dsDNAs. In summary, our results suggest that in addition to mCG sites, unmethylated CA or TG sites also serve as DNA-binding sites for MeCP2 and other MBDcontaining proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. |
Crystal structure of mbd2 complex with methylated cpg island. ➥[+]:CcG/cGG, [-]:cGG/CcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6cnq | transcription-DNA | X-ray (2.151 Å) | Liu K, Xu C, Lei M, Yang A, Loppnau P, Hughes TR, Min J | (2018) "Structural basis for the ability of MBD domains to bind methyl-CG and TG sites in DNA." J. Biol. Chem., 293, 7344-7354. doi: 10.1074/jbc.RA118.001785. | Cytosine methylation is a well characterized epigenetic mark and occurs at both CG and non-CG sites in DNA. Both methylated CG (mCG)- and mCH (H = A, C, or T)-containing DNAs, especially mCAC-containing DNAs, are recognized by methyl-CpG-binding protein 2 (MeCP2) to regulate gene expression in neuron development. However, the molecular mechanism involved in the binding of methyl-CpG-binding domain (MBD) of MeCP2 to these different DNA motifs is unclear. Here, we systematically characterized the DNA-binding selectivity of the MBDs in MeCP2 and MBD1-4 with isothermal titration calorimetry-based binding assays, mutagenesis studies, and X-ray crystallography. We found that the MBD domains of MeCP2 and MBD1-4 bind mCG-containing DNAs independently of the sequence outside the mCG dinucleotide. Moreover, some 1 MBDs bound to both methylated and unmethylated CA dinucleotide- containing DNAs, with a preference for the CAC sequence motif. We also found that MBD domains bind to mCA or nonmethylated CA DNA by recognizing the complementary TG dinucleotide, which is consistent with an overlooked ligand of MeCP2, i.e., the matrix/scaffold attachment regions (MARs/SARs) with a consensus sequence of 5'-GGTGT-3', which was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCAand hmCA-containing dsDNAs. In summary, our results suggest that in addition to mCG sites, unmethylated CA or TG sites also serve as DNA-binding sites for MeCP2 and other MBDcontaining proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. |
Mbd2 in complex with methylated DNA. ➥[+]:AcG/cGT, [-]:cGT/AcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6d1t | DNA binding protein-DNA | X-ray (2.25 Å) | Liu K, Xu C, Tempel W, Arrowsmith CH, Bountra C, Edwards AM, Min J, Structural Genomics Consortium | "Complex of MBD1-MBD and methylated DNA." | Complex of mbd1-mbd and methylated DNA. ➥[+]:AcG/cGT, [-]:cGT/AcG, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 6e93 | DNA binding protein-DNA | X-ray (1.747 Å) | Hudson NO, Whitby FG, Buck-Koehntop BA | (2018) "Structural insights into methylated DNA recognition by the C-terminal zinc fingers of the DNA reader protein ZBTB38." J. Biol. Chem., 293, 19835-19843. doi: 10.1074/jbc.RA118.005147. | Methyl-CpG binding proteins (MBPs) are selective readers of DNA methylation that play an essential role in mediating cellular transcription processes in both normal and diseased cells. This physiological function of MBPs has generated significant interest in understanding the mechanisms by which these proteins read and interpret DNA methylation signals. Zinc finger and BTB domain-containing 38 (ZBTB38) represents one member of the zinc finger (ZF) family of MBPs. We recently demonstrated that the C-terminal ZFs of ZBTB38 exhibit methyl-selective DNA binding within the ((A/G)TmCG(G/A)(mC/T)(G/A)) context both in vitro and within cells. Here we report the crystal structure of the first four C-terminal ZBTB38 ZFs (ZFs 6-9) in complex with the previously identified methylated consensus sequence at 1.75 Å resolution. From the structure, methyl-selective binding is preferentially localized at the 5'-mCpG site of the bound DNA which is facilitated through a series of base-specific interactions from residues within the α-helices of ZF7 and ZF8. ZF6 and ZF9 primarily stabilize ZF7 and ZF8 to facilitate the core base-specific interactions. Further structural and biochemical analyses, including solution NMR spectroscopy and electrophoretic mobility gel shift assays, revealed that the C-terminal ZFs of ZBTB38 utilize an alternative mode of mCpG recognition from the ZF MBPs structurally evaluated to date. Combined these findings provide insight into the mechanism by which this ZF domain of ZBTB38 selectively recognizes methylated CpG sites and expands our understanding for how ZF-containing proteins can interpret this essential epigenetic mark. | Crystal structure of zbtb38 c-terminal zinc fingers 6-9 in complex with methylated DNA. ➥[+]:CcG/cGG, [+]:GcG/cGC, [-]:cGA/TcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, other-contacts |
| 6e94 | DNA binding protein-DNA | X-ray (1.594 Å) | Hudson NO, Whitby FG, Buck-Koehntop BA | (2018) "Structural insights into methylated DNA recognition by the C-terminal zinc fingers of the DNA reader protein ZBTB38." J. Biol. Chem., 293, 19835-19843. doi: 10.1074/jbc.RA118.005147. | Methyl-CpG binding proteins (MBPs) are selective readers of DNA methylation that play an essential role in mediating cellular transcription processes in both normal and diseased cells. This physiological function of MBPs has generated significant interest in understanding the mechanisms by which these proteins read and interpret DNA methylation signals. Zinc finger and BTB domain-containing 38 (ZBTB38) represents one member of the zinc finger (ZF) family of MBPs. We recently demonstrated that the C-terminal ZFs of ZBTB38 exhibit methyl-selective DNA binding within the ((A/G)TmCG(G/A)(mC/T)(G/A)) context both in vitro and within cells. Here we report the crystal structure of the first four C-terminal ZBTB38 ZFs (ZFs 6-9) in complex with the previously identified methylated consensus sequence at 1.75 Å resolution. From the structure, methyl-selective binding is preferentially localized at the 5'-mCpG site of the bound DNA which is facilitated through a series of base-specific interactions from residues within the α-helices of ZF7 and ZF8. ZF6 and ZF9 primarily stabilize ZF7 and ZF8 to facilitate the core base-specific interactions. Further structural and biochemical analyses, including solution NMR spectroscopy and electrophoretic mobility gel shift assays, revealed that the C-terminal ZFs of ZBTB38 utilize an alternative mode of mCpG recognition from the ZF MBPs structurally evaluated to date. Combined these findings provide insight into the mechanism by which this ZF domain of ZBTB38 selectively recognizes methylated CpG sites and expands our understanding for how ZF-containing proteins can interpret this essential epigenetic mark. | Crystal structure of zbtb38 c-terminal zinc fingers 6-9 k1055r in complex with methylated DNA. ➥[+]:CcG/cGG, [+]:GcG/cGC, [-]:cGA/TcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 6jnm | DNA binding protein-DNA | X-ray (2.05 Å) | Qiu Q, Mei H, Deng X, He K, Wu B, Yao Q, Zhang J, Lu F, Ma J, Cao X | (2019) "DNA methylation repels targeting of Arabidopsis REF6." Nat Commun, 10, 2063. doi: 10.1038/s41467-019-10026-1. | RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12), a Jumonji C (JmjC)-domain-containing H3K27me3 histone demethylase, finds its target loci in Arabidopsis genome by directly recognizing the CTCTGYTY motif via its zinc-finger (ZnF) domains. REF6 tends to bind motifs located in active chromatin states that are depleted for heterochromatic modifications. However, the underlying mechanism remains unknown. Here, we show that REF6 preferentially bind to hypo-methylated CTCTGYTY motifs in vivo, and that CHG methylation decreases REF6 DNA binding affinity in vitro. In addition, crystal structures of ZnF-clusters in complex with DNA oligonucleotides reveal that 5-methylcytosine is unfavorable for REF6 binding. In drm1 drm2 cmt2 cmt3 (ddcc) quadruple mutants, in which non-CG methylation is significantly reduced, REF6 can ectopically bind a small number of new target loci, most of which are located in or neighbored with short TEs in euchromatic regions. Collectively, our findings reveal that DNA methylation, likely acting in combination with other epigenetic modifications, may partially explain why REF6 binding is depleted in heterochromatic loci. | Ref6 znf2-4-nac004-mc3 complex. ➥[+]:TcT/AGA, [-]:AGA/TcT, is-WC-paired, is-in-duplex, other-contacts |
| 6jnn | DNA binding protein-DNA | X-ray (2.6 Å) | Qiu Q, Mei H, Deng X, He K, Wu B, Yao Q, Zhang J, Lu F, Ma J, Cao X | (2019) "DNA methylation repels targeting of Arabidopsis REF6." Nat Commun, 10, 2063. doi: 10.1038/s41467-019-10026-1. | RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12), a Jumonji C (JmjC)-domain-containing H3K27me3 histone demethylase, finds its target loci in Arabidopsis genome by directly recognizing the CTCTGYTY motif via its zinc-finger (ZnF) domains. REF6 tends to bind motifs located in active chromatin states that are depleted for heterochromatic modifications. However, the underlying mechanism remains unknown. Here, we show that REF6 preferentially bind to hypo-methylated CTCTGYTY motifs in vivo, and that CHG methylation decreases REF6 DNA binding affinity in vitro. In addition, crystal structures of ZnF-clusters in complex with DNA oligonucleotides reveal that 5-methylcytosine is unfavorable for REF6 binding. In drm1 drm2 cmt2 cmt3 (ddcc) quadruple mutants, in which non-CG methylation is significantly reduced, REF6 can ectopically bind a small number of new target loci, most of which are located in or neighbored with short TEs in euchromatic regions. Collectively, our findings reveal that DNA methylation, likely acting in combination with other epigenetic modifications, may partially explain why REF6 binding is depleted in heterochromatic loci. | Ref6 znf2-4-nac004-mc1 complex. ➥[+]:TcT/AGA, [-]:AGA/TcT, is-WC-paired, is-in-duplex, other-contacts |
| 6jtq | DNA binding protein-DNA | X-ray (2.48 Å) | Liu L, Yi C | "RVD HA specifically contacts 5mC through van der Waals interactions." | Rvd ha specifically contacts 5mc through van der waals interactions. ➥[+]:TcG/CGA, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 6jvz | DNA binding protein-DNA | X-ray (2.48 Å) | Liu L, Zhang Y, Liu M, Wei W, Yi C, Peng J | (2020) "Structural Insights into the Specific Recognition of 5-methylcytosine and 5-hydroxymethylcytosine by TAL Effectors." J.Mol.Biol., 432, 1035-1047. doi: 10.1016/j.jmb.2019.11.023. | Transcription activator-like effectors (TALEs) recognize DNA through repeat-variable diresidues (RVDs), and TALE-DNA interactions are sensitive to DNA modifications. Our previous study deciphered the recognition of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) by TALEs. Here, we report seven crystal structures of TALE-DNA complexes. The 5mC-specific RVD HA recognizes 5mC through van der Waals interactions and exhibits highly similar loop conformation to natural RVDs. The degenerate RVD RG contacts 5mC and 5hmC via van der Waals interactions as well; however, its loop conformation differs significantly. The loop conformations of universal RVD R* and 5hmC-specific RVD Q* are similar to that of RG, while the interactions of R* with C/5mC/5hmC and Q* with 5hmC are mediated by waters. Together, our findings illustrate the molecular basis for the specific recognition of 5mC and 5hmC by multiple noncanonical TALEs and provide insights into the plasticity of the TALE RVD loops. | Rvd ha specifically contacts 5mc through van der waals interactions. ➥[+]:TcG/CGA, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6jw1 | DNA binding protein-DNA | X-ray (2.49 Å) | Liu L, Zhang Y, Liu M, Wei W, Yi C, Peng J | (2020) "Structural Insights into the Specific Recognition of 5-methylcytosine and 5-hydroxymethylcytosine by TAL Effectors." J.Mol.Biol., 432, 1035-1047. doi: 10.1016/j.jmb.2019.11.023. | Transcription activator-like effectors (TALEs) recognize DNA through repeat-variable diresidues (RVDs), and TALE-DNA interactions are sensitive to DNA modifications. Our previous study deciphered the recognition of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) by TALEs. Here, we report seven crystal structures of TALE-DNA complexes. The 5mC-specific RVD HA recognizes 5mC through van der Waals interactions and exhibits highly similar loop conformation to natural RVDs. The degenerate RVD RG contacts 5mC and 5hmC via van der Waals interactions as well; however, its loop conformation differs significantly. The loop conformations of universal RVD R* and 5hmC-specific RVD Q* are similar to that of RG, while the interactions of R* with C/5mC/5hmC and Q* with 5hmC are mediated by waters. Together, our findings illustrate the molecular basis for the specific recognition of 5mC and 5hmC by multiple noncanonical TALEs and provide insights into the plasticity of the TALE RVD loops. | Universal rvd r* accommodates 5mc via water-mediated interactions. ➥[+]:TcG/CGA, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6jw3 | DNA binding protein-DNA | X-ray (3.1 Å) | Liu L, Zhang Y, Liu M, Wei W, Yi C, Peng J | (2020) "Structural Insights into the Specific Recognition of 5-methylcytosine and 5-hydroxymethylcytosine by TAL Effectors." J.Mol.Biol., 432, 1035-1047. doi: 10.1016/j.jmb.2019.11.023. | Transcription activator-like effectors (TALEs) recognize DNA through repeat-variable diresidues (RVDs), and TALE-DNA interactions are sensitive to DNA modifications. Our previous study deciphered the recognition of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) by TALEs. Here, we report seven crystal structures of TALE-DNA complexes. The 5mC-specific RVD HA recognizes 5mC through van der Waals interactions and exhibits highly similar loop conformation to natural RVDs. The degenerate RVD RG contacts 5mC and 5hmC via van der Waals interactions as well; however, its loop conformation differs significantly. The loop conformations of universal RVD R* and 5hmC-specific RVD Q* are similar to that of RG, while the interactions of R* with C/5mC/5hmC and Q* with 5hmC are mediated by waters. Together, our findings illustrate the molecular basis for the specific recognition of 5mC and 5hmC by multiple noncanonical TALEs and provide insights into the plasticity of the TALE RVD loops. | Degenerate rvd rg forms a distinct loop conformation. ➥[+]:TcG/CGA, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6lew | DNA binding protein-DNA | X-ray (2.48 Å) | Liu L, Yi C | "RVD HA specifically contacts 5mC through van der Waals interactions." | Rvd ha specifically contacts 5mc through van der waals interactions. ➥[+]:TcG/CGA, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 6m2v | DNA binding protein-DNA | X-ray (3.0 Å) | Abhishek S, Nakarakanti NK, Deeksha W, Rajakumara E | (2021) "Mechanistic insights into recognition of symmetric methylated cytosines in CpG and non-CpG DNA by UHRF1 SRA." Int.J.Biol.Macromol., 170, 514-522. doi: 10.1016/j.ijbiomac.2020.12.149. | Non-CpG DNA methylation (non-mCpG) is enriched in the genome of brain neurons and germline cells in mammals. Accumulation of non-mCpG during postnatal brain development correlates with gene regulation and inactivation of distal regulatory elements. Recently, UHRF1 has been found to contribute to de novo non-CpG methylation, however, whether UHRF1 could recognize non-mCpG is unknown. Here, we have demonstrated through calorimetric measurements that the UHRF1 SRA can recognize mCpH and fully-mCpHpG, types of non-mCpG. Our ITC binding studies endorse the preferential reading of hemi-mCpG by UHRF1 SRA and also show 6-fold weaker binding for fully-mCpG than hemi-mCpG. Despite presence of symmetrical (5-methyl cytosine) 5mCs, stoichiometry of 1:1 for UHRF1 SRA binding to fully-mCpG indicates that UHRF1 SRA may not form a stable complex with fully-mCpG DNA. Contrarily, UHRF1 SRA recognizes fully-mCpHpG with a stoichiometry of 2:1 protein to DNA duplex with binding affinity higher than fully-mCpG. Our crystal structure of UHRF1 SRA bound to fully-mCpHpG DNA reveals dual flip-out mechanism of 5mC recognition. Metadynamics studies corroborates with ITC data that UHRF1 SRA could not form a stable complex with fully-mCpG DNA. Altogether, this study demonstrates that UHRF1 SRA recognizes non-mCpG DNA and exhibits contrasting mechanisms for hemi-mCpG and fully-mCpHpG DNA recognition. | Crystal structure of uhrf1 sra complexed with fully-mchg DNA.. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 6mg1 | transcription-DNA | X-ray (1.75 Å) | Yang J, Horton JR, Wang D, Ren R, Li J, Sun D, Huang Y, Zhang X, Blumenthal RM, Cheng X | (2019) "Structural basis for effects of CpA modifications on C/EBP beta binding of DNA." Nucleic Acids Res., 47, 1774-1785. doi: 10.1093/nar/gky1264. | CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPβ binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPβ DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPβ. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors. | C-terminal bzip domain of human c-ebpbeta with 16bp methylated oligonucleotide containing consensus recognition sequence-c2 crystal form. ➥[+]:GcA/TGc, [+]:GcG/cGc, [-]:TGc/GcA, [-]:cGc/GcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6mg2 | transcription-DNA | X-ray (1.928 Å) | Yang J, Horton JR, Wang D, Ren R, Li J, Sun D, Huang Y, Zhang X, Blumenthal RM, Cheng X | (2019) "Structural basis for effects of CpA modifications on C/EBP beta binding of DNA." Nucleic Acids Res., 47, 1774-1785. doi: 10.1093/nar/gky1264. | CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPβ binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPβ DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPβ. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors. | C-terminal bzip domain of human c-ebpbeta with 16bp methylated oligonucleotide containing consensus recognition sequence-c2221 crystal form. ➥[+]:GcA/TGc, [+]:GcG/cGc, [-]:TGc/GcA, [-]:cGc/GcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 6mg3 | transcription-DNA | X-ray (2.05 Å) | Yang J, Horton JR, Wang D, Ren R, Li J, Sun D, Huang Y, Zhang X, Blumenthal RM, Cheng X | (2019) "Structural basis for effects of CpA modifications on C/EBP beta binding of DNA." Nucleic Acids Res., 47, 1774-1785. doi: 10.1093/nar/gky1264. | CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPβ binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPβ DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPβ. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors. | V285a mutant of the c-terminal bzip domain of human c-ebpbeta with 16bp methylated oligonucleotide containing consensus recognition sequence. ➥[+]:GcA/TGc, [+]:GcG/cGc, [-]:TGc/GcA, [-]:cGc/GcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 6mht | transferase-DNA | X-ray (2.05 Å) | Kumar S, Horton JR, Jones GD, Walker RT, Roberts RJ, Cheng X | (1997) "DNA containing 4'-thio-2'-deoxycytidine inhibits methylation by HhaI methyltransferase." Nucleic Acids Res., 25, 2773-2783. doi: 10.1093/nar/25.14.2773. | 4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected phosphoramidite compatible with solid phase DNA synthesis. When incorporated as the target cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M. HhaI, methyl transfer was strongly inhibited. In contrast, these same oligonucleotides were normal substrates for the cognate restriction endonuclease R. HhaI and its isoschizomer R. Hin P1I. M. HhaI was able to bind both 4'-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium conditions. However, the presence of 4'-thio-2'-deoxycytidine decreased the half-life of the complex by >10-fold. The crystal structure of a ternary complex of M. HhaI, AdoMet and DNA containing 4'-thio-2'-deoxycytidine was solved at 2.05 A resolution with a crystallographic R-factor of 0.186 and R-free of 0.231. The structure is not grossly different from previously solved ternary complexes containing M. HhaI, DNA and AdoHcy. The difference electron density suggests partial methylation at C5 of the flipped target 4'-thio-2'-deoxycytidine. The inhibitory effect of the 4'sulfur atom on enzymatic activity may be traced to perturbation of a step in the methylation reaction after DNA binding but prior to methyl transfer. This inhibitory effect can be partially overcome after a considerably long time in the crystal environment where the packing prevents complex dissociation and the target is accurately positioned within the active site. | Ternary structure of hhai methyltransferase with adohcy and DNA containing 4'-thio-2'deoxycytidine at the target. ➥[+]:GcC/GGC, hydrophobic-with-AA, is-WC-paired, is-in-duplex |
| 6ml6 | transcription-DNA | X-ray (1.54 Å) | Ren R, Hardikar S, Horton JR, Lu Y, Zeng Y, Singh AK, Lin K, Coletta LD, Shen J, Lin Kong CS, Hashimoto H, Zhang X, Chen T, Cheng X | (2019) "Structural basis of specific DNA binding by the transcription factor ZBTB24." Nucleic Acids Res., 47, 8388-8398. doi: 10.1093/nar/gkz557. | ZBTB24, encoding a protein of the ZBTB family of transcriptional regulators, is one of four known genes-the other three being DNMT3B, CDCA7 and HELLS-that are mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a genetic disorder characterized by DNA hypomethylation and antibody deficiency. The molecular mechanisms by which ZBTB24 regulates gene expression and the biological functions of ZBTB24 are poorly understood. Here, we identified a 12-bp consensus sequence [CT(G/T)CCAGGACCT] occupied by ZBTB24 in the mouse genome. The sequence is present at multiple loci, including the Cdca7 promoter region, and ZBTB24 binding is mostly associated with gene activation. Crystallography and DNA-binding data revealed that the last four of the eight zinc fingers (ZFs) (i.e. ZF5-8) in ZBTB24 confer specificity of DNA binding. Two ICF missense mutations have been identified in the ZBTB24 ZF domain, which alter zinc-binding cysteine residues. We demonstrated that the corresponding C382Y and C407G mutations in mouse ZBTB24 abolish specific DNA binding and fail to induce Cdca7 expression. Our analyses indicate and suggest a structural basis for the sequence specific recognition by a transcription factor centrally important for the pathogenesis of ICF syndrome. | Zbtb24 zinc fingers 4-8 with 19+1mer DNA oligonucleotide (sequence 4 with a cpa 5mc modification). ➥[+]:GcA/TGC, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6ml7 | transcription-DNA | X-ray (1.75 Å) | Ren R, Hardikar S, Horton JR, Lu Y, Zeng Y, Singh AK, Lin K, Coletta LD, Shen J, Lin Kong CS, Hashimoto H, Zhang X, Chen T, Cheng X | (2019) "Structural basis of specific DNA binding by the transcription factor ZBTB24." Nucleic Acids Res., 47, 8388-8398. doi: 10.1093/nar/gkz557. | ZBTB24, encoding a protein of the ZBTB family of transcriptional regulators, is one of four known genes-the other three being DNMT3B, CDCA7 and HELLS-that are mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a genetic disorder characterized by DNA hypomethylation and antibody deficiency. The molecular mechanisms by which ZBTB24 regulates gene expression and the biological functions of ZBTB24 are poorly understood. Here, we identified a 12-bp consensus sequence [CT(G/T)CCAGGACCT] occupied by ZBTB24 in the mouse genome. The sequence is present at multiple loci, including the Cdca7 promoter region, and ZBTB24 binding is mostly associated with gene activation. Crystallography and DNA-binding data revealed that the last four of the eight zinc fingers (ZFs) (i.e. ZF5-8) in ZBTB24 confer specificity of DNA binding. Two ICF missense mutations have been identified in the ZBTB24 ZF domain, which alter zinc-binding cysteine residues. We demonstrated that the corresponding C382Y and C407G mutations in mouse ZBTB24 abolish specific DNA binding and fail to induce Cdca7 expression. Our analyses indicate and suggest a structural basis for the sequence specific recognition by a transcription factor centrally important for the pathogenesis of ICF syndrome. | Zbtb24 zinc fingers 4-8 with 19+1mer DNA oligonucleotide (sequence 4 with a cpg 5mc modification). ➥[+]:AcG/CGT, is-WC-paired, is-in-duplex, other-contacts |
| 6ogk | DNA binding protein-DNA | X-ray (1.65 Å) | Lei M, Tempel W, Chen S, Liu K, Min J | (2019) "Plasticity at the DNA recognition site of the MeCP2 mCG-binding domain." Biochim Biophys Acta Gene Regul Mech, 1862, 194409. doi: 10.1016/j.bbagrm.2019.194409. | MeCP2 is an abundant protein, involved in transcriptional repression by binding to CG and non-CG methylated DNA. However, MeCP2 might also function as a transcription activator as MeCP2 is found bound to sparsely methylated promoters of actively expressed genes. Furthermore, Attachment Region Binding Protein (ARBP), the chicken ortholog of MeCP2, has been reported to bind to Matrix/scaffold attachment regions (MARs/SARs) DNA with an unmethylated 5'-CAC/GTG-3' consensus sequence. In our previous study, although we have systemically measured the binding abilities of MBDs to unmethylated CAC/GTG DNA and the complex structures reveal that the MBD2-MBD (MBD of MBD2) binds to the unmethylated CAC/GTG DNA by recognizing the complementary GTG trinucleotide, how the MeCP2-MBD (MBD of MeCP2) recognizes the unmethylated CAC/GTG DNA, especially the MARs DNA, is still unclear. In this study, we investigated the binding characteristics of MeCP2 in recognizing unmethylated 5'-CAC/GTG-3' motif containing DNA by binding and structural studies. We found that MeCP2-MBD binds to MARs DNA with a comparable binding affinity to mCG DNA, and the MeCP2-CAC/GTG complex structure revealed that MeCP2 residues R111 and R133 form base-specific interactions with the GTG motif. For comparison, we also determined crystal structures of the MeCP2-MBD bound to mCG and mCAC/GTG DNA, respectively. Together, these crystal structures illustrate the adaptability of the MeCP2-MBD toward the GTG motif as well as the mCG DNA, and also provide structural basis of a biological role of MeCP2 as a transcription activator and its disease implications in Rett syndrome. | Mecp2 mbd in complex with DNA. ➥[+]:AcA/TGT, is-WC-paired, is-in-duplex, other-contacts |
| 6r64 | hydrolase | X-ray (2.64 Å) | Slyvka A, Zagorskaite E, Czapinska H, Sasnauskas G, Bochtler M | (2019) "Crystal structure of the EcoKMcrA N-terminal domain (NEco): recognition of modified cytosine bases without flipping." Nucleic Acids Res., 47, 11943-11955. doi: 10.1093/nar/gkz1017. | EcoKMcrA from Escherichia coli restricts CpG methylated or hydroxymethylated DNA, and may act as a barrier against host DNA. The enzyme consists of a novel N-terminal specificity domain that we term NEco, and a C-terminal catalytic HNH domain. Here, we report that NEco and full-length EcoKMcrA specificities are consistent. NEco affinity to DNA increases more from hemi- to full-methylation than from non- to hemi-methylation, indicating cooperative binding of the methyl groups. We determined the crystal structures of NEco in complex with fully modified DNA containing three variants of the Y5mCGR EcoKMcrA target sequence: C5mCGG, T5mCGA and T5hmCGA. The structures explain the specificity for the two central base pairs and one of the flanking pairs. As predicted based on earlier biochemical experiments, NEco does not flip any DNA bases. The proximal and distal methyl groups are accommodated in separate pockets. Changes to either pocket reduce DNA binding by NEco and restriction by EcoKMcrA, confirming the relevance of the crystallographically observed binding mode in solution. | N-terminal domain of modification dependent ecokmcra restriction endonuclease (neco) in complex with c5mcgg target sequence. ➥[+]:CcG/cGG, [-]:cGG/CcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6t21 | hydrolase | X-ray (2.07 Å) | Slyvka A, Zagorskaite E, Czapinska H, Sasnauskas G, Bochtler M | (2019) "Crystal structure of the EcoKMcrA N-terminal domain (NEco): recognition of modified cytosine bases without flipping." Nucleic Acids Res., 47, 11943-11955. doi: 10.1093/nar/gkz1017. | EcoKMcrA from Escherichia coli restricts CpG methylated or hydroxymethylated DNA, and may act as a barrier against host DNA. The enzyme consists of a novel N-terminal specificity domain that we term NEco, and a C-terminal catalytic HNH domain. Here, we report that NEco and full-length EcoKMcrA specificities are consistent. NEco affinity to DNA increases more from hemi- to full-methylation than from non- to hemi-methylation, indicating cooperative binding of the methyl groups. We determined the crystal structures of NEco in complex with fully modified DNA containing three variants of the Y5mCGR EcoKMcrA target sequence: C5mCGG, T5mCGA and T5hmCGA. The structures explain the specificity for the two central base pairs and one of the flanking pairs. As predicted based on earlier biochemical experiments, NEco does not flip any DNA bases. The proximal and distal methyl groups are accommodated in separate pockets. Changes to either pocket reduce DNA binding by NEco and restriction by EcoKMcrA, confirming the relevance of the crystallographically observed binding mode in solution. | N-terminal domain of ecokmcra restriction endonuclease (neco) in complex with t5mcga target sequence. ➥[+]:TcG/cGA, [-]:cGA/TcG, hydrophobic-with-AA, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6w8v | transferase-DNA | X-ray (3.12 Å) | Adam S, Anteneh H, Hornisch M, Wagner V, Lu J, Radde NE, Bashtrykov P, Song J, Jeltsch A | (2020) "DNMT1 activity, base flipping mechanism and genome-wide DNA methylation are regulated by the DNA sequence context." Nat Commun. | Crystal structure of mouse dnmt1 in complex with acg DNA. ➥[+]:Cc./.GG, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 6w8w | transferase-DNA | X-ray (3.0 Å) | Adam S, Anteneh H, Hornisch M, Wagner V, Lu J, Radde NE, Bashtrykov P, Song J, Jeltsch A | (2020) "DNMT1 activity, base flipping mechanism and genome-wide DNA methylation are regulated by the DNA sequence context." Nat Commun. | Crystal structure of mouse dnmt1 in complex with ccg DNA. ➥[+]:Ac./.GT, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 6x6e | DNA binding protein | X-ray (2.0 Å) | Liu X, Weikum ER, Tilo D, Vinson C, Ortlund EA | (2021) "Structural basis for glucocorticoid receptor recognition of both unmodified and methylated binding sites, precursors of a modern recognition element." Nucleic Acids Res., 49, 8923-8933. doi: 10.1093/nar/gkab605. | The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS. | Glucocorticoid receptor DNA binding domain in complex with methylated precursor for a modern recognition element (methylated pre-gbs). ➥[+]:GcG/CGC, [-]:CGG/CcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6x9i | transferase | X-ray (2.2 Å) | Pappalardi MB, Keenan K, Cockerill M, Kellner WA, Stowell A, Sherk C, Wong K, Pathuri S, Briand J, Steidel M, Chapman P, Groy A, Wiseman AK, McHugh CF, Campobasso N, Graves AP, Fairweather E, Werner T, Raoof A, Butlin RJ, Rueda L, Horton JR, Fosbenner DT, Zhang C, Handler JL, Muliaditan M, Mebrahtu M, Jaworski JP, McNulty DE, Burt C, Eberl HC, Taylor AN, Ho T, Merrihew S, Foley SW, Rutkowska A, Li M, Romeril SP, Goldberg K, Zhang X, Kershaw CS, Bantscheff M, Jurewicz AJ, Minthorn E, Grandi P, Patel M, Benowitz AB, Mohammad HP, Gilmartin AG, Prinjha RK, Ogilvie D, Carpenter C, Heerding D, Baylin SB, Jones PA, Cheng X, King BW, Luengo JI, Jordan AM, Waddell I, Kruger RG, McCabe MT | (2021) "Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia." Nat Cancer, 2, 1002-1017. | DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia. | Human dnmt1(729-1600) bound to zebularine-containing 12mer dsDNA. ➥[+]:CcC/GGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 6x9j | transferase-transferase inhibitor-DNA | X-ray (1.79 Å) | Pappalardi MB, Keenan K, Cockerill M, Kellner WA, Stowell A, Sherk C, Wong K, Pathuri S, Briand J, Steidel M, Chapman P, Groy A, Wiseman AK, McHugh CF, Campobasso N, Graves AP, Fairweather E, Werner T, Raoof A, Butlin RJ, Rueda L, Horton JR, Fosbenner DT, Zhang C, Handler JL, Muliaditan M, Mebrahtu M, Jaworski JP, McNulty DE, Burt C, Eberl HC, Taylor AN, Ho T, Merrihew S, Foley SW, Rutkowska A, Li M, Romeril SP, Goldberg K, Zhang X, Kershaw CS, Bantscheff M, Jurewicz AJ, Minthorn E, Grandi P, Patel M, Benowitz AB, Mohammad HP, Gilmartin AG, Prinjha RK, Ogilvie D, Carpenter C, Heerding D, Baylin SB, Jones PA, Cheng X, King BW, Luengo JI, Jordan AM, Waddell I, Kruger RG, McCabe MT | (2021) "Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia." Nat Cancer, 2, 1002-1017. | Human dnmt1(729-1600) bound to zebularine-containing 12mer dsDNA and inhibitor gsk3830052. ➥[+]:CcG/uGG, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 6x9k | transferase-transferase inhibitor-DNA | X-ray (2.65 Å) | Pappalardi MB, Keenan K, Cockerill M, Kellner WA, Stowell A, Sherk C, Wong K, Pathuri S, Briand J, Steidel M, Chapman P, Groy A, Wiseman AK, McHugh CF, Campobasso N, Graves AP, Fairweather E, Werner T, Raoof A, Butlin RJ, Rueda L, Horton JR, Fosbenner DT, Zhang C, Handler JL, Muliaditan M, Mebrahtu M, Jaworski JP, McNulty DE, Burt C, Eberl HC, Taylor AN, Ho T, Merrihew S, Foley SW, Rutkowska A, Li M, Romeril SP, Goldberg K, Zhang X, Kershaw CS, Bantscheff M, Jurewicz AJ, Minthorn E, Grandi P, Patel M, Benowitz AB, Mohammad HP, Gilmartin AG, Prinjha RK, Ogilvie D, Carpenter C, Heerding D, Baylin SB, Jones PA, Cheng X, King BW, Luengo JI, Jordan AM, Waddell I, Kruger RG, McCabe MT | (2021) "Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia." Nat Cancer, 2, 1002-1017. | Human dnmt1(729-1600) bound to zebularine-containing 12mer dsDNA and inhibitor gsk3685032a. ➥[+]:CcG/uGG, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 6ykf | hydrolase | X-ray (1.48 Å) | Pastor M, Czapinska H, Helbrecht I, Krakowska K, Lutz T, Xu SY, Bochtler M | (2021) "Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA." Nucleic Acids Res., 49, 1708-1723. doi: 10.1093/nar/gkaa1218. | Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues. | Vcam4i restriction endonuclease in the presence of 5mc-modified ssDNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 6ymg | hydrolase | X-ray (3.14 Å) | Pastor M, Czapinska H, Helbrecht I, Krakowska K, Lutz T, Xu SY, Bochtler M | (2021) "Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA." Nucleic Acids Res., 49, 1708-1723. doi: 10.1093/nar/gkaa1218. | Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues. | Vcam4i restriction endonuclease in complex with 5mc-modified dsDNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 7cy6 | transferase | X-ray (2.1 Å) | Li W, Zhang T, Sun M, Shi Y, Zhang XJ, Xu GL, Ding J | (2021) "Molecular mechanism for vitamin C-derived C 5 -glyceryl-methylcytosine DNA modification catalyzed by algal TET homologue CMD1." Nat Commun, 12, 744. doi: 10.1038/s41467-021-21061-2. | C5-glyceryl-methylcytosine (5gmC) is a novel DNA modification catalyzed by algal TET homologue CMD1 using vitamin C (VC) as co-substrate. Here, we report the structures of CMD1 in apo form and in complexes with VC or/and dsDNA. CMD1 exhibits comparable binding affinities for DNAs of different lengths, structures, and 5mC levels, and displays a moderate substrate preference for 5mCpG-containing DNA. CMD1 adopts the typical DSBH fold of Fe2+/2-OG-dependent dioxygenases. The lactone form of VC binds to the active site and mono-coordinates the Fe2+ in a manner different from 2-OG. The dsDNA binds to a positively charged cleft of CMD1 and the 5mC/C is inserted into the active site and recognized by CMD1 in a similar manner as the TET proteins. The functions of key residues are validated by mutagenesis and activity assay. Our structural and biochemical data together reveal the molecular mechanism for the VC-derived 5gmC DNA modification by CMD1. | Crystal structure of cmd1 in complex with 5mc-DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 7cy8 | transferase | X-ray (2.4 Å) | Li W, Zhang T, Sun M, Shi Y, Zhang XJ, Xu GL, Ding J | (2021) "Molecular mechanism for vitamin C-derived C 5 -glyceryl-methylcytosine DNA modification catalyzed by algal TET homologue CMD1." Nat Commun, 12, 744. doi: 10.1038/s41467-021-21061-2. | C5-glyceryl-methylcytosine (5gmC) is a novel DNA modification catalyzed by algal TET homologue CMD1 using vitamin C (VC) as co-substrate. Here, we report the structures of CMD1 in apo form and in complexes with VC or/and dsDNA. CMD1 exhibits comparable binding affinities for DNAs of different lengths, structures, and 5mC levels, and displays a moderate substrate preference for 5mCpG-containing DNA. CMD1 adopts the typical DSBH fold of Fe2+/2-OG-dependent dioxygenases. The lactone form of VC binds to the active site and mono-coordinates the Fe2+ in a manner different from 2-OG. The dsDNA binds to a positively charged cleft of CMD1 and the 5mC/C is inserted into the active site and recognized by CMD1 in a similar manner as the TET proteins. The functions of key residues are validated by mutagenesis and activity assay. Our structural and biochemical data together reveal the molecular mechanism for the VC-derived 5gmC DNA modification by CMD1. | Crystal structure of cmd1 in complex with 5mc-DNA and vitamin c. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| 7fef | DNA binding protein-DNA | X-ray (2.39 Å) | Wu Z, Chen S, Zhou M, Jia L, Li Z, Zhang X, Min J, Liu K | (2022) "Family-wide Characterization of Methylated DNA Binding Ability of Arabidopsis MBDs." J.Mol.Biol., 434, 167404. doi: 10.1016/j.jmb.2021.167404. | 13 MBD-containing genes (AtMBD1-13) have been identified in Arabidopsis thaliana so far, however, their DNA binding ability is still controversial. Here, we systematically measured the DNA binding affinities of these MBDs by ITC and EMSA binding assays, except for those of pseudogenes AtMBD3 and AtMBD13, and found that only AtMBD6 and AtMBD7 function as methylated DNA readers. We also found that the MBD of AtMBD5 exhibits very weak binding to methylated DNA compared to that of AtMBD6. To further investigate the structural basis of AtMBDs in binding to methylated DNA, we determined the complex structure of the AtMBD6 MBD with a 12mer mCG DNA and the apo structure of the AtMBD5 MBD. Structural analysis coupled with mutagenesis studies indicated that, in addition to the conserved arginine fingers contributing to the DNA binding specificity, the residues located in the loop1 and α1 are also essential for the methylated DNA binding of these MBDs in Arabidopsis thaliana, which explains why AtMBD5 MBD and the other AtMBDs display very weak or no binding to methylated DNA. Thus, our study here systematically demonstrates the DNA binding ability of the MBDs in Arabidopsis thaliana, which also provides a general guideline in understanding the DNA binding ability of the MBDs in other plants as a whole. | Crystal structure of atmbd6 with DNA. ➥[+]:AcG/cGT, [-]:cGT/AcG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 7mwk | DNA binding protein-DNA | X-ray (2.453 Å) | Liu K, Dong A, Edwards AM, Arrowsmith CH, Min J, Structural Genomics Consortium (SGC) | "Crystal structure of MBD2 with DNA." | Crystal structure of mbd2 with DNA. ➥[+]:AcG/cGT, [-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA | |
| 7mwm | DNA binding protein-DNA | X-ray (1.601 Å) | Liu K, Dong A, Edwards AM, Arrowsmith CH, Min J, Structural Genomics Consortium (SGC) | "Crystal structure of MBD2 with DNA." | Crystal structure of mbd2 with DNA. ➥[+]:AcG/cGT, [-]:cGT/AcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA | |
| 7ne3 | oxidoreductase | X-ray (2.26 Å) | Ravichandran M, Rafalski D, Davies CI, Ortega-Recalde O, Nan X, Glanfield CR, Kotter A, Misztal K, Wang AH, Wojciechowski M, Razew M, Mayyas IM, Kardailsky O, Schwartz U, Zembrzycki K, Morison IM, Helm M, Weichenhan D, Jurkowska RZ, Krueger F, Plass C, Zacharias M, Bochtler M, Hore TA, Jurkowski TP | (2022) "Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function." Sci Adv, 8, eabm2427. doi: 10.1126/sciadv.abm2427. | TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor-binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support. | Human tet2 in complex with favourable DNA substrate.. ➥[-]:.GT/Ac., is-WC-paired, is-in-duplex, not-WC-paired, not-in-duplex, stacking-with-AA |
| 7ne6 | oxidoreductase | X-ray (2.3 Å) | Ravichandran M, Rafalski D, Davies CI, Ortega-Recalde O, Nan X, Glanfield CR, Kotter A, Misztal K, Wang AH, Wojciechowski M, Razew M, Mayyas IM, Kardailsky O, Schwartz U, Zembrzycki K, Morison IM, Helm M, Weichenhan D, Jurkowska RZ, Krueger F, Plass C, Zacharias M, Bochtler M, Hore TA, Jurkowski TP | (2022) "Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function." Sci Adv, 8, eabm2427. doi: 10.1126/sciadv.abm2427. | TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor-binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support. | Human tet2 in complex with unfavourable DNA substrate.. ➥[-]:.GC/Gc., is-WC-paired, is-in-duplex, not-WC-paired, not-in-duplex, stacking-with-AA |
| 7nx5 | viral protein | X-ray (2.5 Å) | Bernaudat F, Gustems M, Gunther J, Oliva MF, Buschle A, Gobel C, Pagniez P, Lupo J, Signor L, Muller CW, Morand P, Sattler M, Hammerschmidt W, Petosa C | (2022) "Structural basis of DNA methylation-dependent site selectivity of the Epstein-Barr virus lytic switch protein ZEBRA/Zta/BZLF1." Nucleic Acids Res., 50, 490-511. doi: 10.1093/nar/gkab1183. | In infected cells, Epstein-Barr virus (EBV) alternates between latency and lytic replication. The viral bZIP transcription factor ZEBRA (Zta, BZLF1) regulates this cycle by binding to two classes of ZEBRA response elements (ZREs): CpG-free motifs resembling the consensus AP-1 site recognized by cellular bZIP proteins and CpG-containing motifs that are selectively bound by ZEBRA upon cytosine methylation. We report structural and mutational analysis of ZEBRA bound to a CpG-methylated ZRE (meZRE) from a viral lytic promoter. ZEBRA recognizes the CpG methylation marks through a ZEBRA-specific serine and a methylcytosine-arginine-guanine triad resembling that found in canonical methyl-CpG binding proteins. ZEBRA preferentially binds the meZRE over the AP-1 site but mutating the ZEBRA-specific serine to alanine inverts this selectivity and abrogates viral replication. Our findings elucidate a DNA methylation-dependent switch in ZEBRA's transactivation function that enables ZEBRA to bind AP-1 sites and promote viral latency early during infection and subsequently, under appropriate conditions, to trigger EBV lytic replication by binding meZREs. | Crystal structure of the epstein-barr virus protein zebra (bzlf1, zta) bound to a methylated DNA duplex. ➥[+]:TcG/cGA, [-]:cGC/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 7r77 | transferase-DNA | cryo-EM (3.0 Å) | Wang J, Catania S, Wang C, de la Cruz MJ, Rao B, Madhani HD, Patel DJ | (2022) "Structural insights into DNMT5-mediated ATP-dependent high-fidelity epigenome maintenance." Mol.Cell, 82, 1186-1198.e6. doi: 10.1016/j.molcel.2022.01.028. | Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance type cytosine methyltransferase identified in the fungal or protist kingdoms, the first dependent on adenosine triphosphate (ATP), and the most hemimethyl-DNA-specific enzyme known. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA binding first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out of the DNA duplex. ATP binding then triggers striking structural reconfigurations of the methyltransferase catalytic pocket to enable cofactor binding, completion of base flipping, and catalysis. Bound unmethylated DNA does not open the catalytic pocket and is instead ejected upon ATP binding, driving high fidelity. This unprecedented chaperone-like, enzyme-remodeling role of the SNF2 ATPase domain illuminates how energy is used to enable faithful epigenetic memory. | cryo-EM structure of dnmt5 binary complex with hemimethylated DNA. ➥[+]:GcC/GGC, is-WC-paired, is-in-duplex, other-contacts |
| 7r78 | transferase-DNA | cryo-EM (3.5 Å) | Wang J, Catania S, Wang C, de la Cruz MJ, Rao B, Madhani HD, Patel DJ | (2022) "Structural insights into DNMT5-mediated ATP-dependent high-fidelity epigenome maintenance." Mol.Cell, 82, 1186-1198.e6. doi: 10.1016/j.molcel.2022.01.028. | Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance type cytosine methyltransferase identified in the fungal or protist kingdoms, the first dependent on adenosine triphosphate (ATP), and the most hemimethyl-DNA-specific enzyme known. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA binding first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out of the DNA duplex. ATP binding then triggers striking structural reconfigurations of the methyltransferase catalytic pocket to enable cofactor binding, completion of base flipping, and catalysis. Bound unmethylated DNA does not open the catalytic pocket and is instead ejected upon ATP binding, driving high fidelity. This unprecedented chaperone-like, enzyme-remodeling role of the SNF2 ATPase domain illuminates how energy is used to enable faithful epigenetic memory. | cryo-EM structure of dnmt5 quaternary complex with hemimethylated DNA, amp-pnp and sah. ➥[+]:Gc./.GC, is-WC-paired, is-in-duplex, other-contacts |
| 7ray | DNA binding protein-DNA | X-ray (1.78 Å) | Liu K, Dong A, Loppnau P, Edwards AM, Arrowsmith CH, Min J, Structural Genomics Consortium (SGC) | "Crystal structure of MBD2 with DNA." | Crystal structure of mbd2 with DNA. ➥[+]:AcG/cGT, [-]:cGT/AcG, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 7sfc | transferase-transferase inhibitor | X-ray (1.97 Å) | Horton JR, Pathuri S, Wong K, Ren R, Rueda L, Fosbenner DT, Heerding DA, McCabe MT, Pappalardi MB, Zhang X, King BW, Cheng X | (2022) "Structural characterization of dicyanopyridine containing DNMT1-selective, non-nucleoside inhibitors." Structure, 30, 793-802.e5. doi: 10.1016/j.str.2022.03.009. | DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1. | Human dnmt1(729-1600) bound to zebularine-containing 12mer dsDNA and inhibitor gsk3735967a. ➥[+]:CcG/uGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 7sfe | transferase-transferase inhibitor | X-ray (2.55 Å) | Horton JR, Pathuri S, Wong K, Ren R, Rueda L, Fosbenner DT, Heerding DA, McCabe MT, Pappalardi MB, Zhang X, King BW, Cheng X | (2022) "Structural characterization of dicyanopyridine containing DNMT1-selective, non-nucleoside inhibitors." Structure, 30, 793-802.e5. doi: 10.1016/j.str.2022.03.009. | DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1. | Human dnmt1(729-1600) bound to zebularine-containing 12mer dsDNA and inhibitor gsk3830334a. ➥[+]:CcG/uGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 7sff | transferase-transferase inhibitor | X-ray (2.05 Å) | Horton JR, Pathuri S, Wong K, Ren R, Rueda L, Fosbenner DT, Heerding DA, McCabe MT, Pappalardi MB, Zhang X, King BW, Cheng X | (2022) "Structural characterization of dicyanopyridine containing DNMT1-selective, non-nucleoside inhibitors." Structure, 30, 793-802.e5. doi: 10.1016/j.str.2022.03.009. | DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1. | Human dnmt1(729-1600) bound to zebularine-containing 12mer dsDNA and inhibitor gsk3852279b. ➥[+]:CcG/uGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 7sfg | transferase-transferase inhibitor | X-ray (2.43 Å) | Horton JR, Pathuri S, Wong K, Ren R, Rueda L, Fosbenner DT, Heerding DA, McCabe MT, Pappalardi MB, Zhang X, King BW, Cheng X | (2022) "Structural characterization of dicyanopyridine containing DNMT1-selective, non-nucleoside inhibitors." Structure, 30, 793-802.e5. doi: 10.1016/j.str.2022.03.009. | DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1. | Human dnmt1(729-1600) bound to zebularine-containing 12mer dsDNA and cofactor sam. ➥[+]:CcC/GGG, is-WC-paired, is-in-duplex, stacking-with-AA |
| 7t02 | transferase-DNA | cryo-EM (3.8 Å) | Wang J, Catania S, Wang C, de la Cruz MJ, Rao B, Madhani HD, Patel DJ | (2022) "Structural insights into DNMT5-mediated ATP-dependent high-fidelity epigenome maintenance." Mol.Cell, 82, 1186-1198.e6. doi: 10.1016/j.molcel.2022.01.028. | Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance type cytosine methyltransferase identified in the fungal or protist kingdoms, the first dependent on adenosine triphosphate (ATP), and the most hemimethyl-DNA-specific enzyme known. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA binding first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out of the DNA duplex. ATP binding then triggers striking structural reconfigurations of the methyltransferase catalytic pocket to enable cofactor binding, completion of base flipping, and catalysis. Bound unmethylated DNA does not open the catalytic pocket and is instead ejected upon ATP binding, driving high fidelity. This unprecedented chaperone-like, enzyme-remodeling role of the SNF2 ATPase domain illuminates how energy is used to enable faithful epigenetic memory. | cryo-EM structure of dnmt5 pseudo-ternary complex solved by incubation with hemimethylated DNA and sam. ➥[+]:Gc./.GC, is-WC-paired, is-in-duplex, other-contacts |
| 7yhp | DNA binding protein-DNA | cryo-EM (3.1 Å) | Du X, Yang Z, Xie G, Wang C, Zhang L, Yan K, Yang M, Li S, Zhu JK, Du J | (2023) "Molecular basis of the plant ROS1-mediated active DNA demethylation." Nat.Plants, 9, 271-279. doi: 10.1038/s41477-022-01322-8. | Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly excise 5-methyl-cytosine (5mC), representing an efficient DNA demethylation pathway distinct from that of animals. Here, we report the cryo-electron microscopy structures of an Arabidopsis ROS1 catalytic fragment in complex with substrate DNA, mismatch DNA and reaction intermediate, respectively. The substrate 5mC is flipped-out from the DNA duplex and subsequently recognized by the ROS1 base-binding pocket through hydrophobic and hydrogen-bonding interactions towards the 5-methyl group and Watson-Crick edge respectively, while the different protonation states of the bases determine the substrate preference for 5mC over T:G mismatch. Together with the structure of the reaction intermediate complex, our structural and biochemical studies revealed the molecular basis for substrate specificity, as well as the reaction mechanism underlying 5mC demethylation by the ROS1/DME family of plant-specific DNA demethylases. | Cryoem structure of arabidopsis ros1 in complex with 5mc-dsDNA at 3.1 angstroms resolution. ➥hydrophobic-with-AA, not-WC-paired, not-in-duplex |
| 7yun | DNA binding protein | X-ray (2.13 Å) | Liu K, Zhang J, Xiao Y, Yang A, Song X, Li Y, Chen Y, Hughes TR, Min J | (2023) "Structural insights into DNA recognition by the BEN domain of the transcription factor BANP." J.Biol.Chem., 299, 104734. doi: 10.1016/j.jbc.2023.104734. | The BEN domain-containing transcription factors regulate transcription by recruiting chromatin-modifying factors to specific chromatin regions via their DNA-binding BEN domains. The BEN domain of BANP has been shown to bind to a CGCG DNA sequence or an AAA-containing MARs (matrix attachment regions) DNA sequence. Consistent with these in vivo observations, we identified an optimal DNA binding sequence of AAATCTCG by PBM (protein binding microarray), which was also confirmed by our ITC (Isothermal Titration Calorimetry) and mutagenesis results to uncover additional mechanistic details about DNA binding by the BEN domain of BANP. We then determined crystal structures of the BANP BEN domain in apo form and in complex with a CGCG-containing DNA, respectively, which revealed that the BANP BEN domain mainly used the electrostatic interactions to bind DNA with some base-specific interactions with the TC motifs. Our ITC results also showed that BANP bound to unmethylated and methylated DNAs with comparable binding affinities. Our complex structure of BANP-mCGCG revealed that the BANP BEN domain bound to the unmethylated and methylated DNAs in a similar mode and cytosine methylation did not get involved in binding, which is also consistent with our observations from the complex structures of the BEND6 BEN domain with the CGCG or CGmCG DNAs. Taken together, our results further elucidate the elements important for DNA recognition and transcriptional regulation by the BANP BEN domain-containing transcription factor. | Crystal structure of human bend6 ben domain in complex with methylated DNA. ➥[+]:GcG/CGc, [-]:CGc/GcG, is-WC-paired, is-in-duplex, other-contacts, stacking-with-AA |
| 8c56 | transferase | X-ray (2.4 Å) | Wojciechowski M, Czapinska H, Krwawicz J, Rafalski D, Bochtler M | "Cytosine analogues as DNA methyltransferase substrates and inhibitors." | Cpg specific m.mpei methyltransferase crystallized in the presence of 2'-deoxy-5-methylzebularine (5mz) and 5-methylcytosine containing dsDNA. ➥[-]:GGC/GcC, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 8c57 | transferase | X-ray (1.95 Å) | Wojciechowski M, Czapinska H, Krwawicz J, Rafalski D, Bochtler M | "Cytosine analogues as DNA methyltransferase substrates and inhibitors." | Cpg specific m.mpei methyltransferase crystallized in the presence of 5,6-dihydro-5-azacytosine (converted to 5m-dhac) and 5-methylcytosine containing dsDNA. ➥[-]:GGC/GcC, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 8c58 | transferase | X-ray (1.85 Å) | Wojciechowski M, Czapinska H, Krwawicz J, Rafalski D, Bochtler M | "Cytosine analogues as DNA methyltransferase substrates and inhibitors." | Cpg specific m.mpei methyltransferase crystallized in the presence of 5-hydroxycytosine and 5-methylcytosine containing dsDNA. ➥[-]:GGC/GcC, is-WC-paired, is-in-duplex, stacking-with-AA | |
| 8c59 | transferase | X-ray (1.7 Å) | Wojciechowski M, Czapinska H, Krwawicz J, Rafalski D, Bochtler M | "Cytosine analogues as DNA methyltransferase substrates and inhibitors." | Cpg specific m.mpei methyltransferase crystallized in the presence of 5-bromocytosine (converted to 5mc) and 5-methylcytosine containing dsDNA. ➥[-]:GGC/GcC, hydrophobic-with-AA, is-WC-paired, is-in-duplex, not-WC-paired, not-in-duplex, stacking-with-AA | |
| 8eng | transcription-DNA | X-ray (1.25 Å) | Terrell JR, Taylor SJ, Schneider AL, Lu Y, Vernon TN, Xhani S, Gumpper RH, Luo M, Wilson WD, Steidl U, Poon GMK | (2023) "DNA selection by the master transcription factor PU.1." Cell Rep, 42, 112671. doi: 10.1016/j.celrep.2023.112671. | The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5'-GGAA-3') flanked by variable sequences, affinity is negotiated by direct readout on the 5' flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3' flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1's characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins. | Human pu.1 ets-domain (165-270) bound to d(aataagcggaagtggg) with hemi-methylated cpg (forward strand). ➥[+]:GcG/CGC, is-WC-paired, is-in-duplex, other-contacts |
| 8eo1 | transcription-DNA | X-ray (1.28 Å) | Terrell JR, Taylor SJ, Schneider AL, Lu Y, Vernon TN, Xhani S, Gumpper RH, Luo M, Wilson WD, Steidl U, Poon GMK | (2023) "DNA selection by the master transcription factor PU.1." Cell Rep, 42, 112671. doi: 10.1016/j.celrep.2023.112671. | The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5'-GGAA-3') flanked by variable sequences, affinity is negotiated by direct readout on the 5' flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3' flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1's characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins. | Human pu.1 ets-domain (165-270) bound to d(aataagcggaagtggg) with hemi-methylated cpg (reverse strand). ➥[-]:CGG/CcG, is-WC-paired, is-in-duplex, other-contacts |
| 8eo4 | transcription-DNA | X-ray (1.24 Å) | Terrell JR, Taylor SJ, Schneider AL, Lu Y, Vernon TN, Xhani S, Gumpper RH, Luo M, Wilson WD, Steidl U, Poon GMK | (2023) "DNA selection by the master transcription factor PU.1." Cell Rep, 42, 112671. doi: 10.1016/j.celrep.2023.112671. | The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5'-GGAA-3') flanked by variable sequences, affinity is negotiated by direct readout on the 5' flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3' flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1's characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins. | Human pu.1 ets-domain (165-270) bound to d(aataagcggaagtggg) with di-methylated cpg sites. ➥[+]:GcG/cGC, [-]:cGG/CcG, is-WC-paired, is-in-duplex, other-contacts |
| 8htx | DNA binding protein | X-ray (2.8 Å) | Liu K, Zhang J, Xiao Y, Yang A, Song X, Li Y, Chen Y, Hughes TR, Min J | (2023) "Structural insights into DNA recognition by the BEN domain of the transcription factor BANP." J.Biol.Chem., 299, 104734. doi: 10.1016/j.jbc.2023.104734. | The BEN domain-containing transcription factors regulate transcription by recruiting chromatin-modifying factors to specific chromatin regions via their DNA-binding BEN domains. The BEN domain of BANP has been shown to bind to a CGCG DNA sequence or an AAA-containing MARs (matrix attachment regions) DNA sequence. Consistent with these in vivo observations, we identified an optimal DNA binding sequence of AAATCTCG by PBM (protein binding microarray), which was also confirmed by our ITC (Isothermal Titration Calorimetry) and mutagenesis results to uncover additional mechanistic details about DNA binding by the BEN domain of BANP. We then determined crystal structures of the BANP BEN domain in apo form and in complex with a CGCG-containing DNA, respectively, which revealed that the BANP BEN domain mainly used the electrostatic interactions to bind DNA with some base-specific interactions with the TC motifs. Our ITC results also showed that BANP bound to unmethylated and methylated DNAs with comparable binding affinities. Our complex structure of BANP-mCGCG revealed that the BANP BEN domain bound to the unmethylated and methylated DNAs in a similar mode and cytosine methylation did not get involved in binding, which is also consistent with our observations from the complex structures of the BEND6 BEN domain with the CGCG or CGmCG DNAs. Taken together, our results further elucidate the elements important for DNA recognition and transcriptional regulation by the BANP BEN domain-containing transcription factor. | Crystal structure of banp in complex with methylated DNA. ➥[+]:TcG/CGA, [-]:CGA/TcG, is-WC-paired, is-in-duplex, other-contacts |
| 8ik4 | DNA binding protein | X-ray (2.1 Å) | Adhav VA, Saikrishnan K | "Structural basis of target recognition by the DNA binding domain of McrBC." | Structure of DNA binding domain of mcrbc endonuclease bound to hemimethylated DNA: l68f mutant. ➥not-WC-paired, not-in-duplex, stacking-with-AA | |
| 8jkk | DNA binding protein-DNA | X-ray (2.3 Å) | Zhang L, Mu Y, Li T, Hu J, Lin H, Zhang L | (2024) "Molecular basis of an atypical dsDNA 5mC/6mA bifunctional dioxygenase CcTet from Coprinopsis cinerea in catalyzing dsDNA 5mC demethylation." Nucleic Acids Res. doi: 10.1093/nar/gkae066. | The eukaryotic epigenetic modifications 5-methyldeoxycytosine (5mC) and N6-methyldeoxyadenine (6mA) have indispensable regulatory roles in gene expression and embryonic development. We recently identified an atypical bifunctional dioxygenase CcTet from Coprinopsis cinerea that works on both 5mC and 6mA demethylation. The nonconserved residues Gly331 and Asp337 of CcTet facilitate 6mA accommodation, while D337F unexpectedly abolishes 5mC oxidation activity without interfering 6mA demethylation, indicating a prominent distinct but unclear 5mC oxidation mechanism to the conventional Tet enzymes. Here, we assessed the molecular mechanism of CcTet in catalyzing 5mC oxidation by representing the crystal structure of CcTet-5mC-dsDNA complex. We identified the distinct mechanism by which CcTet recognizes 5mC-dsDNA compared to 6mA-dsDNA substrate. Moreover, Asp337 was found to have a central role in compensating for the loss of a critical 5mC-stablizing H-bond observed in conventional Tet enzymes, and stabilizes 5mC and subsequent intermediates through an H-bond with the N4 atom of the substrates. These findings improve our understanding of Tet enzyme functions in the dsDNA 5mC and 6mA demethylation pathways, and provide useful information for future discovery of small molecular probes targeting Tet enzymes in DNA active demethylation processes. | Crystal structure of the dioxygenase cctet from coprinopsis cinerea bound to 12bp 5-methylcytosine (5mc) containing duplex DNA. ➥not-WC-paired, not-in-duplex, stacking-with-AA |
| pdb id | class | method | authors | reference | abstract | annotation |