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June 30th, 2025
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Institute of Chemistry, Martin Luther-University Halle-Wittenberg, Weinbergweg 22, Halle (Saale) 06120, Germany
molecular biology
biorxiv

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

Volkenandt, S.Open in Google Scholar•Belyaeva, J.Open in Google Scholar•Li, T.Open in Google Scholar•Elgeti, M.Open in Google Scholar•Metzler, R.Open in Google Scholar•Imhof, P.Open in Google Scholar•C. Williams, D.Open in Google Scholar•Bagherpoor Helabad, M.Open in Google Scholar

Methyl-CpG binding domain 2 (MBD2) is a critical epigenetic regulator that recognizes and binds with high selectivity to methylated CpG dinucleotides, a fundamental epigenetic mark involved in gene regulation and chromatin organization. Therefore, understanding the interactions and conformational dynamics that drive MBD2\'s high selectivity and strong binding affinity to methylated CpG is crucial to unraveling its regulatory mechanisms. In this study, through extensive classical MD simulations, we investigated the formation of the methylated CpG-MBD2 recognition complex. By positioning MBD2 one base pair downstream of its target mCpG site, we observed its transition to a stable complex at the target site within the microsecond timescale. Significantly, we observe that upon binding to methylated CpG, MBD2 forms two distinct stable complexes, with one state adopting a conformation that agrees well with the X-ray structure of the complex (primary state) and the other exhibiting a distinct binding conformation with lower affinity for methylated CpG (secondary state). Our data indicate that S189 serves as a key macro-switch, where loss of its interaction with the methylcytosine backbone shifts the complex toward the secondary state. This is supported by MD simulations of the S189A mutation, where we observe that the complex adopts a conformation closer to the secondary state. Ultimately, our NMR experiments confirm that the S189A mutation does not alter MBD2\'s selectivity for methylated CpG, while fluorescence polarization demonstrates a reduction in binding affinity, consistent with our MD simulations. Together, these findings strongly suggest that MBD2 adopts a bistable equilibrium in binding to methylated CpG dinucleotides.

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