Structural and Functional Consequences of Arg512 Mutations in Methyl-CpG-Binding Domain Protein 4

Methyl-CpG-binding domain protein 4 (MBD4) is a base excision repair glycosylase that removes the thymine of a T:G mispair. If left unrepaired, these mismatches can cause C>T transitions. Human MBD4 uses an arginine finger, R468, to flip the mispaired thymine into its active site and to stabilize the orphaned guanine. MBD4 also possesses a second highly conserved arginine, R512, in close proximity to R468, whose role remains unknown despite an R512W mutation catalogued in colorectal cancers and an R512Q mutation reported in Myelodysplastic Syndromes. Here, we investigate the structural and functional significance of R512 using MBD4 variants R512A, R512K, R512Q, and R512W. We find that the native hydrogen-bonding network mediated by R512 is critical for active-site stability, and that R512K is the only variant to retain catalytic activity. This mutant preserves the wild-type rate of base excision despite a reduced affinity for the DNA substrate. Structural characterization of R512K MBD4 bound to T:G-mismatched DNA reveals an ordered water molecule in this pocket. This water molecule, along with the lysine amino group, retain 6 of the 7 native R512 hydrogen bonds, thereby preserving the local architecture of the enzyme. Comparative structural modeling of the catalytically inactive R512A, R512Q, and R512W variants suggests that loss of localized positive charge combined with disruption of the hydrogen-bonding network accounts for their inactivity. Together, these findings establish an essential role for R512 in catalytic activity and suggest how mutation of this residue can disrupt the BER pathway and contribute to tumorigenesis.