Histone lysine methylation (Kme) encodes essential information modulating many biological processes including gene expression and transcriptional regulation. However, the atomic-level recognition mechanisms of methylated histones by their respective adaptor proteins are still elusive. For instance, it is unclear how L3MBTL1, a methyl-lysine histone code reader, recognizes equally well both mono- and di-methyl marks, but ignores unmodified and trimethylated lysine residues. We made use of Molecular Dynamics (MD) and Free Energy Perturbation (FEP) techniques in order to investigate the energetics and dynamics of the methyllysine recognition. Isothermal Titration Calorimetry (ITC) was employed to experimentally validate the computational findings. Both computational and experimental methods were applied to a set of designed “biophysical” probes that mimic the shape of a single lysine residue and reproduce the binding affinities of cognate histone peptides. Our results suggest that, besides forming favorable interactions, the L3MBTL1 binding pocket energetically penalizes both methylation states and has most probably evolved as a “compromise” that non-optimally fit to both mono- and di-methyl-lysine marks
