Solution structure of the methyl-CpG-binding domain of the methylation-dependent transcriptional repressor MBD1

CPG methylation in vertebrates is important for gene silencing, alterations in chromatin structure and genomic stability, and differences in the DNA-m ethylation status are correlated with imprinting phenomena, carcinogenesis and embryonic development. Methylation signals are interpreted by protein f actors that contain shared methyl-CpG-binding domains (MBDs). We have deter mined the solution structure of the MBD of the human methylation-dependent transcriptional repressor MBD1 by multi-dimensional heteronuclear NR;IR spe ctroscopy. It folds into an alpha/beta-sandwich structure with characterist ic loops. Basic residues conserved in the MBD family are largely confined t o one face of this fold and a flexible loop, which together form a large po sitively charged surface. Site-directed mutagenesis and chemical shift chan ges upon complexing with a methylated DNA facilitated identification of thi s surface as the DNA interaction site. In addition to three basic residues, conserved Tyr34 and Asp32 were shown to be important for the DNA binding.