Molecular dynamics study of the recognition of dimethylated CpG sites by MBD1 protein

The cell is able to regulate which genes to express via chemical marks on the DNA and on the histone proteins. In all vertebrates, the modification on the DNA is methylation at position 5 of the two cytosines present in the dinucleotide sequence CpG. The information encoded by these chemical marks on the DNA is processed by a family of protein factors containing a conserved methyl-CpG binding domain (MBD). Essential to their function, the MBD proteins are able to bind DNA containing dimethylated CpG sites, whereas binding to unmethylated sites is not observed. In this paper, we perform molecular dynamics simulations to investigate the mechanism by which the mCpG binding domain of MBD1 is able to bind specifically dimethylated CpG sites. We find that the binding affinity of MBD1 to a DNA containing dimethylated CpG site is stronger by 26.4 kJ/mol relative to binding the same DNA but with an unmethylated CpG site. The contribution of each of the methyl groups to the change in free energy is very similar and additive. Therefore, this binding affinity (to a dimethylated DNA) is halved when considered relative to binding a hemimethylated DNA, a result that is also supported by experimental observations. Despite their equal contributions, the two methyl groups are recognized differently by MBD1. In one case, demethylation induces conformational changes in which the hydrophobic patch formed by the conserved residues Val20, Arg22, and Tyr34 moves away from the (methyl)cytosine, weakening the DNA-protein interactions. This is accompanied by an intrusion of a bulk water into the binding site at the protein-DNA interface. As a consequence, there is a reduction and rearrangements of the protein-DNA hydrogen bonds including a loss of a crucial hydrogen bond between Tyr34 and the (methyl)cytosine. The methylcytosine on the opposite strand is recognized by conformational changes of the surrounding conserved hydrophobic residues, Arg44 and Ser45, in which Arg44 participate in the 5mC-Arg-G triad. More specifically, the hydrogens of the methyl group form weak hydrogen bonds with the guanidino group and backbone carbonyl of the conserved Arg44, interactions that are absent when the cytosine is unmethylated. The results presented in this paper contribute to our knowledge of the different ways the chemical mark on the DNA is recognized by the epigenetic machinery.