Unraveling the Origins of Changing Product Specificity Properties of Arginine Methyltransferase PRMT7 by the E181D and E181D/Q329A Mutations through QM/MM MD and Free-Energy Simulations.

Arginine methylations can regulate important biological processes and affect many cellular activities, and the enzymes that catalyze the methylations are protein arginine methyltransferases (PRMTs). The biological consequences of arginine methylations depend on the methylation states of arginine that are determined by the PRMT's product specificity. Nevertheless, it is still unclear how different PRMTs may generate different methylation states for the target proteins. PRMT7 is the only known member of type III PRMT that produces monomethyl arginine (MMA) product. Interestingly, its E181D and E181D/Q329A mutants can catalyze, respectively, the formation of asymmetrically dimethylated arginine (ADMA) and symmetrically dimethylated arginine (SDMA). The reasons as to why the mutants have the abilities to add the second methyl group and E181D (E181D/Q329A) has the unique product specificity in generating ADMA (SDMA) have not been understood. Here, quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) and potential of mean force (PMF) free-energy simulations are performed for the E181D and E181D/Q329A mutants to understand the origin for their ability to generate, respectively, ADMA and SDMA. The simulations show that the free-energy barrier for adding the second methyl group to MMA in E181D (E181D/Q329A) to produce ADMA (SDMA) is considerably lower than the corresponding barriers in wild type and E181D/Q329A (wild type and E181D), consistent with experimental observations. Some important factors that contribute to the change of the activity and product specificity due to the E181D and E181D/Q329A mutations are identified based on the data from the simulations and analysis. It is shown that the transferable methyl group (from SAM) and N (the nitrogen atom that is methylated in the substrate MMA) can only form good near-attack conformations in the E181D reaction state for the methyl transfer (not in wild type and E181D/Q329A), while the transferable methyl group and N (the nitrogen atom that is not methylated in the substrate MMA) can only form good near-attack conformations in E181D/Q329A (not in wild type and E181D). The results suggest that the steric repulsions in the reaction state between the methyl group on MMA and active-site residues (e.g., Q329) and the release of such repulsions (e.g., from the Q329A mutation) may play an important role in generating specific near-attack conformations for the methyl transfer and controlling the product specificity for the mutants. The general principle identified in this work for PRMT7 is expected to be useful for understanding the activity and product specificity of other PRMTs as well.