Structural and Energetic Origin of Different Product Specificities and Activities for SETD3 and Its Mutants on the Methylation of the β‑Actin H73K Peptide: Insights from a QM/MM Study

The methylation of the lysine residue can affect some fundamental biological processes, and specific biological effects of the methylations are often related to product specificity of methyltransferases. The question remains concerning how active-site structural features and dynamics control the activity as well as the number (1, 2, or 3) of methyl groups on methyl lysine products. SET domain containing protein 3 (SETD3) has been identified recently as the β-actin histidine73-N3 methyltransferase, and also, it has a weak methylation activity on the H73K β-actin peptide for which the target H73 residue is mutated into K73. Interestingly, the K73 methylation activity of SETD3 increases significantly as a result of the N255 → A or N255 → F/W273 → A mutation, and the N255A product specificity also differs from that of wild-type. Here, we performed QM/MM molecular dynamics and potential of mean force (PMF) simulations for SETD3 and its mutants (N255A and N255F/W273A) to study how SETD3 and its mutants could have different product specificities and activities for the K73 methylation. The PMF simulations show that the barrier for the first methylation of K73 is higher compared to the barrier of the H73 methylation in SETD3. Moreover, the second methylation of K73 has been found to have a barrier from the free energy simulation that is higher by 2.2 kcal/mol compared to the barrier of the first methyl transfer to K73, agreeing with the suggestion that SETD3 is a monomethylase. For the first, second, and third methylations of K73 in the N255A mutant, the barriers obtained from the PMF simulations for transferring the second and third methyl groups are found to be lower relative to the barrier for the first methyl transfer. Thus, N255A can be considered as a trimethyl lysine methyltransferase. In addition, for the first K73 methylation, the activities from the PMF simulations follow the order of N255F/W273A > N255A > WT, in agreement with experiments. The examination of the structural and dynamic results at the active sites provides better understanding of different product specificities and activities for the K73 methylations in SETD3 and its mutants. It is demonstrated that the existence of well-balanced interactions at the active site leading to the near attack conformation is of crucial importance for the efficient methyl transfers. Moreover, the presence of potential interactions (e.g., the C–H···O and cation−π interactions) that are strengthening at the transition state can also be important. Furthermore, the activity as well as product specificity of the K73 methylation also seems to be controlled by certain active-site water molecules which may be released to provide extra space for the addition of more methyl groups on K73