Exploring the bistable equilibrium of methylated CpG DNA recognition by the MBD2 protein

Methyl-CpG binding domain 2 (MBD2) is a critical epigenetic regulator that selectively binds methylated CpG dinucleotides, key marks controlling gene regulation and chromatin organization. Understanding the interactions and conformational dynamics underlying this high selectivity is essential to elucidate MBD2’s regulatory role. Here, using extensive classical MD simulations totaling over 277 µ s, we explored the formation of the MBD2–mCpG recognition complex. By initially positioning MBD2 one base pair downstream of its target, we observed its transition to the target site within microseconds. Notably, upon binding, MBD2 adopts two distinct stable conformations: a primary state closely resembling the X-ray crystal structure, and a secondary state of reduced affinity that nevertheless retains comparable selectivity for mCpG. Our results establish S189 as a key macro-switch; loss of its interaction with the methylcytosine backbone shifts the equilibrium toward the secondary state. This is corroborated by MD simulations of the S189A mutant, which preferentially adopts the secondary state-like conformation. Complementary NMR experiments confirm that S189A mutation does not alter mCpG selectivity, while fluorescence polarization measurements reveals a reduced binding affinity, consistent with our MD simulations results. Together, these findings indicate that MBD2 binding to methylated CpG involves a bistable equilibrium, providing new insights into how high affinity and adaptability are balanced in epigenetic recognition. In a broader context, our findings suggest that such alternative bound-state equilibria may represent an inherent feature of specific protein–DNA complexes.