DNA recognition by the methyl-CpG binding domain of MeCP2

The methyl-CpG binding domain (MBD) of the transcriptional repressor MeCP2 has been proposed to recognize a single symmetrically methylated CpG base p air via hydrophobic patches on an otherwise positively charged DNA binding surface. We have tested this binding model by analysis of mutant derivative s of the MeCP2 MBD in electrophoretic mobility shift assays complemented by NMR structural analysis. Exposed arginine side chains on the binding face, in particular Arg-lll, were found to be critical for binding. Arg-111 was found to interact with the conserved aspartate side chain Asp-121, which is proposed to orientate the arginine side chain to allow specific contacts w ith the DNA. The conformational flexibility of the disordered B-C loop regi on, which forms part of the binding face, was also shown to be important. I n contrast, mutation off the exposed hydrophobic side chains had a less sev ere effect on DNA binding. This suggests that the Arg-lll side chain may co ntribute to sequence-specific recognition of the CpG site rather than simpl y making nonspecific contacts with the phosphate backbone. The majority of missense mutations within the MBD found in the human genetic disorder Rett syndrome were shown or predicted to affect folding of the domain rather tha n the DNA recognition event directly.