QM/MM MD and free energy simulations of G9a-like protein (GLP) and its mutants: understanding the factors that determine the product specificity.

Certain lysine residues on histone tails could be methylated by protein lysine methyltransferases (PKMTs) using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Since the methylation states of the target lysines play a fundamental role in the regulation of chromatin structure and gene expression, it is important to study the property of PKMTs that allows a specific number of methyl groups (one, two or three) to be added (termed as product specificity). It has been shown that the product specificity of PKMTs may be controlled in part by the existence of specific residues at the active site. One of the best examples is a Phe/Tyr switch found in many PKMTs. Here quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed on wild type G9a-like protein (GLP) and its F1209Y and Y1124F mutants for understanding the energetic origin of the product specificity and the reasons for the change of product specificity as a result of single-residue mutations at the Phe/Tyr switch as well as other positions. The free energy barriers of the methyl transfer processes calculated from our simulations are consistent with experimental data, supporting the suggestion that the relative free energy barriers may determine, at least in part, the product specificity of PKMTs. The changes of the free energy barriers as a result of the mutations are also discussed based on the structural information obtained from the simulations. The results suggest that the space and active-site interactions around the ε-amino group of the target lysine available for methyl addition appear to among the key structural factors in controlling the product specificity and activity of PKMTs.