The solution structure of the domain from MeCP2 that binds to methylated DNA.

MeCP2 is an abundant mammalian protein that binds methylated CpG (mCpG) sequences within double-stranded DNA, represses transcription by recruiting histone deacetylases, and is essential for embryonic development. It is one of a family of proteins which mediate the biological consequences of DNA methylation. These proteins each possess a sequence motif of about 70 residues which, in MeCP2, form a domain necessary and sufficient for binding to mCpG. The solution structure of the mCpG-binding domain (MBD) from MeCP2 has been solved and the DNA-binding surface of the domain mapped using NMR spectroscopy. Residues 95-162 of MeCP2 adopt a novel fold forming a wedge-shaped structure. An N-terminal four-stranded antiparallel beta-sheet forms one face of the wedge, while the other face is formed mainly by a C-terminal helical region. The thin end of the wedge is extended by a long loop between beta-strands B and C containing many basic residues. The B-C loop together with residues in strands B, C and D, and at the N terminus of the alpha-helix, appears to form an interface with methylated DNA. Unstructured residues at the NH2 terminus of the domain are also involved in formation of the complex. The presence of numerous arginine and lysine side-chains on the DNA-binding surface of MBD is consistent with the requirement for the mCpG site to be flanked by non-specific sequences of base-pairs. The absence of symmetry in the domain implies that recognition does not exploit the symmetry of the binding site. A conserved hydrophobic pocket containing the side-chains of Tyr123 and Ile125 on the positively charged beta-sheet face is a candidate for the region of contact with the methyl-groups of the modified cytosine residues.

[1]  A. Bird.  CpG-rich islands and the function of DNA methylation , 1986, Nature.

[2]  A. Bird.  The relationship of DNA methylation to cancer. , 1996, Cancer surveys.

[3]  N. Seeman,et al.  Sequence-specific Recognition of Double Helical Nucleic Acids by Proteins (base Pairs/hydrogen Bonding/recognition Fidelity/ion Binding) , 2022 .

[4]  E. Golemis,et al.  MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[6]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.

[7]  D. Moras,et al.  Effect of cytosine methylation on DNA-DNA recognition at CpG steps. , 1997, Journal of molecular biology.

[8]  A. Bird,et al.  Gene number, noise reduction and biological complexity. , 1995, Trends in genetics : TIG.

[9]  A. Bird,et al.  DNA methylation specifies chromosomal localization of MeCP2 , 1996, Molecular and cellular biology.

[10]  Ad Bax,et al.  Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins , 1993, Journal of biomolecular NMR.

[11]  A. Bird,et al.  The methyl-CpG binding protein MeCP2 is essential for embryonic development in the mouse , 1996, Nature Genetics.

[12]  S. Cross,et al.  Purification of CpG islands using a methylated DNA binding column , 1994, Nature Genetics.

[13]  John W. R. Schwabe,et al.  The crystal structure of a two zinc-finger peptide reveals an extension to the rules for zinc-finger/DNA recognition , 1993, Nature.

[14]  Rudolf Jaenisch,et al.  Targeted mutation of the DNA methyltransferase gene results in embryonic lethality , 1992, Cell.

[15]  D. Barlow,et al.  Multiple roles for DNA methylation in gametic imprinting. , 1996, Current opinion in genetics & development.

[16]  N. Pavletich,et al.  Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A , 1991, Science.

[17]  A. Riggs,et al.  X-chromosome inactivation and cell memory. , 1992, Trends in genetics : TIG.

[18]  A. Bird,et al.  Number of CpG islands and genes in human and mouse. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[19]  L. Kay,et al.  Gradient-Enhanced Triple-Resonance Three-Dimensional NMR Experiments with Improved Sensitivity , 1994 .

[20]  D. Wishart,et al.  The 13C Chemical-Shift Index: A simple method for the identification of protein secondary structure using 13C chemical-shift data , 1994, Journal of biomolecular NMR.

[21]  T. Logan,et al.  A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments , 1993, Journal of biomolecular NMR.

[22]  A. Bird,et al.  Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins , 1998, Molecular and Cellular Biology.

[23]  A. Bird,et al.  MeCP2 Is a Transcriptional Repressor with Abundant Binding Sites in Genomic Chromatin , 1997, Cell.

[24]  S. Kass,et al.  Inactive chromatin spreads from a focus of methylation , 1993, Molecular and cellular biology.

[25]  Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs , 1989, Cell.

[26]  L. Kay,et al.  A Gradient-Enhanced HCCH-TOCSY Experiment for Recording Side-Chain 1H and 13C Correlations in H2O Samples of Proteins , 1993 .

[27]  A. Razin,et al.  DNA methylation and genomic imprinting , 1994, Cell.

[28]  Masashi Suzuki,et al.  A novel mode of DNA recognition by a β‐sheet revealed by the solution structure of the GCC‐box binding domain in complex with DNA , 1998, The EMBO journal.

[29]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[30]  R. Clubb,et al.  NMR structure of the Tn916 integrase–DNA complex , 1999, Nature Structural Biology.

[31]  M. Szyf,et al.  A mammalian protein with specific demethylase activity for mCpG DNA , 1999, Nature.

[32]  Barry L. Stoddard,et al.  DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI , 1998, Nature.

[33]  A. Bird,et al.  Purification, sequence, and cellular localization of a novel chromosomal protein that binds to Methylated DNA , 1992, Cell.

[34]  P. Kraulis,et al.  Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy. , 1994, Biochemistry.

[35]  A. Graessmann,et al.  Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Adrian Bird,et al.  DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein , 1991, Cell.

[37]  M. Bartolomei,et al.  Genomic imprinting in mammals. , 1997, Annual review of genetics.

[38]  A. Bird,et al.  Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. , 1993, Nucleic acids research.

[39]  P. J. Kraulis,et al.  ANSIG: A program for the assignment of protein 1H 2D NMR spectra by interactive computer graphics , 1989, Journal of Magnetic Resonance (1969).

[40]  Rudolf Jaenisch,et al.  Role for DNA methylation in genomic imprinting , 1993, Nature.

[41]  L. Kay,et al.  Enhanced-Sensitivity Triple-Resonance Spectroscopy with Minimal H2O Saturation , 1994 .

[42]  J. Strouboulis,et al.  Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription , 1998, Nature Genetics.

[43]  H Oschkinat,et al.  Automated NOESY interpretation with ambiguous distance restraints: the refined NMR solution structure of the pleckstrin homology domain from beta-spectrin. , 1997, Journal of molecular biology.

[44]  S. Grzesiek,et al.  Measurement of HN-Hα J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods , 1994, Journal of biomolecular NMR.

[45]  Colin A. Johnson,et al.  Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex , 1998, Nature.

[46]  H. Cedar.  DNA methylation and gene activity , 1988, Cell.

[47]  S. Grzesiek,et al.  Correlation of Backbone Amide and Aliphatic Side-Chain Resonances in 13C/15N-Enriched Proteins by Isotropic Mixing of 13C Magnetization , 1993 .

[48]  S. Grzesiek,et al.  Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein , 1992 .