Toggle column:    pdb_id · class · method · authors · reference · abstract · annotation
pdb_id class method authors reference abstract annotation
3C2I transcription regulator X-ray (2.5 Å) Ho, K.L., McNae, I.W., Schmiedeberg, L., Klose, R.J., Bird, A.P., Walkinshaw, M.D. (2008) "MeCP2 binding to DNA depends upon hydration at methyl-CpG." Mol.Cell, 29, 525-531. 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. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:AcG/cGT, [-]:cGG/CcG
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. 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. ➥ 2 5mC groups: stacking-with-AA, not-WC-paired, not-in-duplex
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. 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. ➥ 1 5mC group: stacking-with-AA, not-WC-paired, not-in-duplex
4F6N DNA binding protein-DNA X-ray (2.8 Å) Buck-Koehntop, B.A., Stanfield, R.L., Ekiert, D.C., Martinez-Yamout, M.A., Dyson, H.J., Wilson, I.A., Wright, P.E. (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. 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. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/cGG, other-contacts, [+]:GcG/cGc, [-]:cGT/AcG, [-]:cGc/GcG
4GJP transcription-DNA X-ray (1.94 Å) Yan, N., Deng, D., Yan, C.Y., Yin, P., Pan, X.J., Shi, Y.G. "Crystal structure of a protein complex." To be Published   Crystal structure of the tal effector dhax3 bound to dsDNA containing repetitive methyl-cpg. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:TcG/CGA, [+]:GcG/CGC
4GJR transcription-DNA X-ray (1.85 Å) Yan, N., Deng, D., Yan, C.Y., Yin, P., Pan, X.J., Shi, Y.G. "Crystal structure of a protein complex." To be Published   Crystal structure of the tal effector dhax3 bound to methylated dsDNA. ➥ 6 5mC groups: hydrophobic-with-AA, is-WC-paired, is-in-duplex, [+]:TcT/AGA, stacking-with-AA, [+]:AcC/GGT, [+]:CcC/GGG
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. 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. ➥ 2 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [+]:CcG/cGG, stacking-with-AA, [-]:cGC/GcG
4HP1 DNA binding protein-DNA X-ray (2.25 Å) Xu, Y., Xu, C., Kato, A., Tempel, W., Abreu, J.G., Bian, C., Hu, Y., Hu, D., Zhao, B., Cerovina, T., Diao, J., Wu, F., He, H.H., Cui, Q., Clark, E., Ma, C., Barbara, A., Veenstra, G.J., Xu, G., Kaiser, U.B., Liu, X.S., Sugrue, S.P., He, X., Min, J., Kato, Y., Shi, Y.G. (2012) "Tet3 CXXC Domain and Dioxygenase Activity Cooperatively Regulate Key Genes for Xenopus Eye and Neural Development." Cell, 151, 1200-1213. 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. ➥ 2 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [+]:CcG/cGG, [-]:cGG/CcG
4M9E transcription-DNA X-ray (1.85 Å) Liu, Y., Olanrewaju, Y.O., Zheng, Y., Hashimoto, H., Blumenthal, R.M., Zhang, X., Cheng, X. (2014) "Structural basis for Klf4 recognition of methylated DNA." Nucleic Acids Res., 42, 4859-4867. 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. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, other-contacts, [-]:cGT/AcG
4M9V transcription-DNA X-ray (0.97 Å) Liu, Y., Olanrewaju, Y.O., 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. 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. ➥ 2 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [+]:CcG/cGG
4QEN transcription-DNA X-ray (2.0 Å) Du, J., Johnson, L.M., Groth, M., Feng, S., Hale, C.J., Li, S., Vashisht, A.A., Gallego-Bartolome, J., Wohlschlegel, J.A., Patel, D.J., Jacobsen, S.E. (2014) "Mechanism of DNA Methylation-Directed Histone Methylation by KRYPTONITE." Mol.Cell, 55, 495-504. 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. ➥ 1 5mC group: stacking-with-AA, not-WC-paired, not-in-duplex
4QEO transcription-DNA X-ray (2.0 Å) Du, J., Johnson, L.M., Groth, M., Feng, S., Hale, C.J., Li, S., Vashisht, A.A., Gallego-Bartolome, J., Wohlschlegel, J.A., Patel, D.J., Jacobsen, S.E. (2014) "Mechanism of DNA Methylation-Directed Histone Methylation by KRYPTONITE." Mol.Cell, 55, 495-504. 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. ➥ 1 5mC group: stacking-with-AA, not-WC-paired, not-in-duplex
4R2A DNA binding protein-DNA X-ray (1.59 Å) Hashimoto, H., Olanrewaju, Y.O., Zheng, Y., Wilson, G.G., Zhang, X., Cheng, X. (2014) "Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence." Genes Dev., 28, 2304-2313. In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5 mC), 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5 fC), and 5-carboxylcytosine (5 caC). 5 mC is a major epigenetic signal that acts to regulate gene expression. 5 hmC, 5 fC, and 5 caC 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 5 mC was present and much reduced affinity when 5 hmC or 5 fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5 caC 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 5 caC, 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. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, other-contacts, [-]:cGT/AcG
4R2E DNA binding protein-DNA X-ray (1.84 Å) Hashimoto, H., Olanrewaju, Y.O., Zheng, Y., Wilson, G.G., Zhang, X., Cheng, X. (2014) "Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence." Genes Dev., 28, 2304-2313. In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5 mC), 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5 fC), and 5-carboxylcytosine (5 caC). 5 mC is a major epigenetic signal that acts to regulate gene expression. 5 hmC, 5 fC, and 5 caC 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 5 mC was present and much reduced affinity when 5 hmC or 5 fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5 caC 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 5 caC, 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. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, other-contacts, [-]:cGT/AcG
4R2R DNA binding protein-DNA X-ray (2.09 Å) Hashimoto, H., Olanrewaju, Y.O., Zheng, Y., Wilson, G.G., Zhang, X., Cheng, X. (2014) "Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence." Genes Dev., 28, 2304-2313. In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5 mC), 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5 fC), and 5-carboxylcytosine (5 caC). 5 mC is a major epigenetic signal that acts to regulate gene expression. 5 hmC, 5 fC, and 5 caC 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 5 mC was present and much reduced affinity when 5 hmC or 5 fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5 caC 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 5 caC, 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. ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [-]:cGT/AcG
4R2S DNA binding protein-DNA X-ray (2.49 Å) Hashimoto, H., Olanrewaju, Y.O., Zheng, Y., Wilson, G.G., Zhang, X., Cheng, X. (2014) "Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence." Genes Dev., 28, 2304-2313. In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5 mC), 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5 fC), and 5-carboxylcytosine (5 caC). 5 mC is a major epigenetic signal that acts to regulate gene expression. 5 hmC, 5 fC, and 5 caC 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 5 mC was present and much reduced affinity when 5 hmC or 5 fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5 caC 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 5 caC, 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. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, other-contacts, [-]:cGT/AcG
4X9J transcription regulator-DNA X-ray (1.41 Å) Zandarashvili, L., White, M.A., 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. 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. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, other-contacts, [-]:cGT/AcG
5BT2 DNA binding protein-DNA X-ray (2.2 Å) Chia, J.Y., Tan, W.S., Ng, C.L., Hu, N.J., Foo, H.L., Ho, K.L. (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, 31210-31210.   Mecp2 mbd domain (a140v) in complex with methylated DNA. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:AcG/cGT, [-]:cGG/CcG
5CPK structural protein-DNA X-ray (2.63 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 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. ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [+]:TcG/cGA
5EF6 transcription X-ray (3.0 Å) Yin, Y., Morgunova, E., Jolma, A., Kaasinen, E., Sahu, B., Khund-Sayeed, S., Das, P.K., Kivioja, T., Dave, K., Zhong, F., Nitta, K.R., Taipale, M., Popov, A., Ginno, P.A., 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 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. ➥ 8 5mC groups: hydrophobic-with-AA, is-WC-paired, is-in-duplex, [+]:AcG/cGT, [-]:cGA/TcG, [-]:cGG/CcG
5EGO transcription X-ray (2.54 Å) Yin, Y., Morgunova, E., Jolma, A., Kaasinen, E., Sahu, B., Khund-Sayeed, S., Das, P.K., Kivioja, T., Dave, K., Zhong, F., Nitta, K.R., Taipale, M., Popov, A., Ginno, P.A., 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 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. ➥ 2 5mC groups: hydrophobic-with-AA, is-WC-paired, is-in-duplex, [+]:AcG/cGT, [-]:cGA/TcG
5EMC transcription-DNA X-ray (2.3 Å) Jin, J., Lian, T., SU, X.D. "The effects of cytosine methylation on general transcription factors." To Be Published   Transcription factor grdbd and smgre complex. ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [-]:TGT/AcA
5KE7 transcription-DNA X-ray (2.06 Å) Hashimoto, H., Wang, D., Steves, A.N., Jin, P., Blumenthal, R.M., Zhang, X., Cheng, X. (2016) "Distinctive Klf4 mutants determine preference for DNA methylation status." Nucleic Acids Res., 44, 10177-10185. 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. ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [-]:TGT/AcA
5KE8 transcription-DNA X-ray (2.45 Å) Hashimoto, H., Wang, D., Steves, A.N., Jin, P., Blumenthal, R.M., Zhang, X., Cheng, X. (2016) "Distinctive Klf4 mutants determine preference for DNA methylation status." Nucleic Acids Res., 44, 10177-10185. 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. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, other-contacts, [-]:cGT/AcG
5KL4 transcription-DNA X-ray (1.78 Å) Hashimoto, H., Zhang, X., Zheng, Y., Wilson, G.G., 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. 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. ➥ 2 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [-]:cGT/AcG
5KL5 transcription-DNA X-ray (2.29 Å) Hashimoto, H., Zhang, X., Zheng, Y., Wilson, G.G., 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. 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. ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [-]:cGT/AcG
5KL7 transcription-DNA X-ray (1.58 Å) Hashimoto, H., Zhang, X., Zheng, Y., Wilson, G.G., 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. 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. ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [-]:cGT/AcG
5LTY transcription X-ray (2.66 Å) Yin, Y., Morgunova, E., Jolma, A., Kaasinen, E., Sahu, B., Khund-Sayeed, S., Das, P.K., Kivioja, T., Dave, K., Zhong, F., Nitta, K.R., Taipale, M., Popov, A., Ginno, P.A., 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 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. ➥ 2 5mC groups: hydrophobic-with-AA, is-WC-paired, is-in-duplex, [+]:AcG/cGT
5MCV transcription X-ray (1.6 Å) Golovenko, D., Brauning, B., Vyas, P., Haran, T.E., Rozenberg, H., Shakked, Z. (2018) "New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins." Structure, 26, 1237-1250.e6. 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). ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:gcG/Cgc, [-]:Cgc/gcG
5MCW transcription X-ray (1.9 Å) Golovenko, D., Brauning, B., Vyas, P., Haran, T.E., Rozenberg, H., Shakked, Z. (2018) "New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins." Structure, 26, 1237-1250.e6. 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). ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:gcG/Cgc, [-]:Cgc/gcG
5SZX transcription regulator-DNA X-ray (2.25 Å) Hong, S., Wang, D., Horton, J.R., Zhang, X., Speck, S.H., Blumenthal, R.M., 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. Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5΄- GAG CA-3΄ or 5΄- GAG CA-3΄ (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5΄ end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5΄- GAG C A-3΄) and meZRE2 (5΄- GAG G A-3΄), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cβ atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors. Epstein-barr virus zta DNA binding domain homodimer in complex with methylated DNA. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:TcG/cGA, other-contacts, [-]:cGC/GcG
5T00 transcription regulator-DNA X-ray (2.19 Å) Hashimoto, H., Wang, D., Horton, J.R., Zhang, X., Corces, V.G., Cheng, X. (2017) "Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA." Mol. Cell, 66, 711-720.e3. 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. ➥ 4 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [+]:GcG/cGC, stacking-with-AA, [-]:cGC/GcG
5T01 transcription regulator-DNA X-ray (1.89 Å) Hong, S., Wang, D., Horton, J.R., Zhang, X., Speck, S.H., Blumenthal, R.M., 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. Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5΄- GAG CA-3΄ or 5΄- GAG CA-3΄ (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5΄ end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5΄- GAG C A-3΄) and meZRE2 (5΄- GAG G A-3΄), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cβ atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors. Human c-jun DNA binding domain homodimer in complex with methylated DNA. ➥ 1 5mC group: hydrophobic-with-AA, is-WC-paired, is-in-duplex, [-]:CGT/AcG
5VMU transcription-DNA X-ray (2.35 Å) Nikolova, E.N., Stanfield, R.L., Dyson, H.J., Wright, P.E. (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. 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). ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/cGG, other-contacts, [+]:GcG/cGc, [-]:cGA/TcG, [-]:cGc/GcG
5VMV transcription-DNA X-ray (2.31 Å) Nikolova, E.N., Stanfield, R.L., Dyson, H.J., Wright, P.E. (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. 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. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:TcG/cGA, other-contacts, [+]:GcG/cGc, [-]:cGA/TcG, [-]:cGc/GcG
5VMW transcription-DNA X-ray (2.4 Å) Nikolova, E.N., Stanfield, R.L., Dyson, H.J., Wright, P.E. (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. 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). ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/cGG, other-contacts, [+]:GcG/cGc, [-]:cGA/TcG, [-]:cGc/GcG
5VMX transcription-DNA X-ray (2.05 Å) Nikolova, E.N., Stanfield, R.L., Dyson, H.J., Wright, P.E. (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. 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). ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/CGG, other-contacts, [+]:GcG/CGC
5VMY transcription-DNA X-ray (2.0 Å) Nikolova, E.N., Stanfield, R.L., Dyson, H.J., Wright, P.E. (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. 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). ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/CGG, other-contacts, [+]:GcG/CGC
5VMZ transcription-DNA X-ray (2.32 Å) Nikolova, E.N., Stanfield, R.L., Dyson, H.J., Wright, P.E. (2018) "CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso." Biochemistry, 57, 2109-2120. 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). ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/cGG, other-contacts, [+]:GcG/cGc, [-]:cGA/TcG, [-]:cGc/GcG
6A5N gene regulation-DNA X-ray (2.4 Å) Li, X., Harris, C.J., Zhong, Z., Chen, W., Liu, R., Jia, B., Wang, Z., Li, S., Jacobsen, S.E., 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. 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. ➥ 1 5mC group: stacking-with-AA, not-WC-paired, not-in-duplex
6C1A DNA binding protein-DNA X-ray (2.05 Å) Liu, K., Xu, C., Lei, M., Yang, A., Loppnau, P., Hughes, T.R., 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. 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 selectivities of the MBD domains 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 identity outside the mCG dinucleotide. Moreover, some MBD domains bound to both methylated and unmethylated CA dinucleotide-containing DNAs, with a preference for the CAC sequence motif. We also found that the 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' that was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCA- and 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 MBD-containing proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. Mbd2 in complex with methylated DNA. ➥ 2 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [-]:TGT/AcA
6C1T DNA binding protein-DNA X-ray (1.84 Å) Liu, K., Xu, C., Lei, M., Yang, A., Loppnau, P., Hughes, T.R., 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. 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 selectivities of the MBD domains 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 identity outside the mCG dinucleotide. Moreover, some MBD domains bound to both methylated and unmethylated CA dinucleotide-containing DNAs, with a preference for the CAC sequence motif. We also found that the 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' that was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCA- and 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 MBD-containing proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. Mbd2 in complex with a partially methylated DNA. ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [+]:AcA/TGT
6C1U DNA binding protein-DNA X-ray (2.3 Å) Liu, K., Xu, C., Lei, M., Yang, A., Loppnau, P., Hughes, T.R., 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. 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 selectivities of the MBD domains 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 identity outside the mCG dinucleotide. Moreover, some MBD domains bound to both methylated and unmethylated CA dinucleotide-containing DNAs, with a preference for the CAC sequence motif. We also found that the 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' that was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCA- and 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 MBD-containing proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. Mbd2 in complex with a deoxy-oligonucleotide. ➥ 2 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [-]:TGT/AcA
6C1Y DNA binding protein-DNA X-ray (2.3 Å) Liu, K., Bian, C., Tempel, W., Wernimont, A.K., Arrowsmith, C.H., Bountra, C., Edwards, A.M., Min, J., Structural Genomics Consortium "mbd of human mecp2 in complex with methylated DNA." to be published   Mbd of human mecp2 in complex with methylated DNA. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/cGG, [-]:cGG/CcG
6C2F DNA binding protein-DNA X-ray (2.65 Å) Liu, K., Xu, C., Tempel, W., Arrowsmith, C.H., Bountra, C., Edwards, A.M., Min, J., Structural Genomics Consortium "MBD2 in complex with methylated DNA." to be published   Mbd2 in complex with methylated DNA. ➥ 6 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/CGG
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. 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. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:AcG/cGT, [-]:cGT/AcG
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. 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. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, [-]:cGC/GcG
6CEU transcription-DNA X-ray (2.01 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. 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. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, [-]:cGT/AcG
6CNP transcription-DNA X-ray (2.1 Å) Liu, K., Xu, C., Lei, M., Yang, A., Loppnau, P., Hughes, T.R., 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. 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 selectivities of the MBD domains 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 identity outside the mCG dinucleotide. Moreover, some MBD domains bound to both methylated and unmethylated CA dinucleotide-containing DNAs, with a preference for the CAC sequence motif. We also found that the 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' that was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCA- and 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 MBD-containing proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. Crystal structure of mbd2 complex with methylated cpg island. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:CcG/cGG, [-]:cGG/CcG
6CNQ transcription-DNA X-ray (2.15 Å) Liu, K., Xu, C., Lei, M., Yang, A., Loppnau, P., Hughes, T.R., 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. 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 selectivities of the MBD domains 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 identity outside the mCG dinucleotide. Moreover, some MBD domains bound to both methylated and unmethylated CA dinucleotide-containing DNAs, with a preference for the CAC sequence motif. We also found that the 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' that was identified in early 1990s. Our results also explain why MeCP2 exhibits similar binding affinity to both mCA- and 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 MBD-containing proteins. This discovery expands the genome-wide activity of MBD-containing proteins in gene regulation. Mbd2 in complex with methylated DNA. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:AcG/cGT, [-]:cGT/AcG
6D1T DNA binding protein-DNA X-ray (2.25 Å) Liu, K., Xu, C., Tempel, W., Arrowsmith, C.H., Bountra, C., Edwards, A.M., Min, J., Structural Genomics Consortium "Complex of MBD1-MBD and methylated DNA." to be published   Complex of mbd1-mbd and methylated DNA. ➥ 2 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:AcG/cGT, [-]:cGT/AcG
6E93 DNA binding protein-DNA X-ray (1.75 Å) Hudson, N.O., Whitby, F.G., Buck-Koehntop, B.A. (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. 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 of 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. ➥ 3 5mC groups: hydrophobic-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, [+]:CcG/cGG, other-contacts, [-]:cGA/TcG
6E94 DNA binding protein-DNA X-ray (1.59 Å) Hudson, N.O., Whitby, F.G., Buck-Koehntop, B.A. (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. 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 of 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. ➥ 3 5mC groups: hydrophobic-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGC, stacking-with-AA, [+]:CcG/cGG, other-contacts, [-]:cGA/TcG
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, 2063-2063. 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. ➥ 2 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [+]:TcT/AGA, [-]:AGA/TcT
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, 2063-2063. 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. ➥ 4 5mC groups: other-contacts, is-WC-paired, is-in-duplex, [-]:AGA/TcT, [+]:TcT/AGA
6MG2 transcription-DNA X-ray (1.93 Å) Yang, J., Horton, J.R., Wang, D., Ren, R., Li, J., Sun, D., Huang, Y., Zhang, X., Blumenthal, R.M., Cheng, X. (2019) "Structural basis for effects of CpA modifications on C/EBP beta binding of DNA." Nucleic Acids Res., 47, 1774-1785. 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. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGc, hydrophobic-with-AA, [+]:GcA/TGc, other-contacts, [-]:cGc/GcG, [-]:TGc/GcA
6MG3 transcription-DNA X-ray (2.05 Å) Yang, J., Horton, J.R., Wang, D., Ren, R., Li, J., Sun, D., Huang, Y., Zhang, X., Blumenthal, R.M., Cheng, X. (2019) "Structural basis for effects of CpA modifications on C/EBP beta binding of DNA." Nucleic Acids Res., 47, 1774-1785. 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. ➥ 4 5mC groups: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcG/cGc, hydrophobic-with-AA, [+]:GcA/TGc, other-contacts, [-]:cGc/GcG, [-]:TGc/GcA
6ML6 transcription-DNA X-ray (1.54 Å) Ren, R., Hardikar, S., Horton, J.R., Lu, Y., Zeng, Y., Singh, A.K., Lin, K., Coletta, L.D., Shen, J., Shuet, C., Kong, L., Hashimoto, H., Zhang, X., Chen, T., Cheng, X. (2019) "Structural basis of specific DNA binding by the transcription factor ZBTB24." Nucleic Acids Res. 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). ➥ 1 5mC group: stacking-with-AA, is-WC-paired, is-in-duplex, [+]:GcA/TGC
6ML7 transcription-DNA X-ray (1.75 Å) Ren, R., Hardikar, S., Horton, J.R., Lu, Y., Zeng, Y., Singh, A.K., Lin, K., Coletta, L.D., Shen, J., Shuet, C., Kong, L., Hashimoto, H., Zhang, X., Chen, T., Cheng, X. (2019) "Structural basis of specific DNA binding by the transcription factor ZBTB24." Nucleic Acids Res. 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). ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [+]:AcG/CGT
6OGK DNA binding protein-DNA X-ray (1.65 Å) Lei, M., Tempel, W., Arrowsmith, C.H., Bountra, C., Edwards, A.M., Min, J. "MeCP2 MBD in complex with DNA." To Be Published   Mecp2 mbd in complex with DNA. ➥ 1 5mC group: other-contacts, is-WC-paired, is-in-duplex, [+]:AcA/TGT
pdb_id class method authors reference abstract annotation