Theoretical Study On Catalytic Mechanism And Product Specificity Of Protein Arginine Methyltransferase PRMT7

As one of the important types of post-translational modifications of proteins,arginine methylations can regulate a variety of biological processes and affect various cell activities.The enzymes that catalyze arginine methylations are called protein arginine methyltransferases(PRMTs).The biological consequences of arginine methylations depend on the methylation states of arginine on the substrates,which are determined by the product specificities of PRMTs.The PRMTs family of enzymes can be divided into three types according to the methylation states of products they generate.Type I PRMTs can catalyze the formation of asymmetric dimethylarginine(?-N^G,N^G dimethylarginine or ADMA)and mono-methylarginine(MMA)products;Type II PRMTs can catalyze the formation of symmetrical dimethyl arginine(?-N^G,N'^G-dimethylarginine or SDMA)and MMA products;Protein arginine methyltransferase 7(PRMT7)is the only Type III PRMT that solely produces MMA products.Many diseases like cancer metastasis,diseases associated with DNA damage,pluripotency,and parasitic infection are associated with the activity of PRMT7.Interestingly,experimental studies have found that the E181D and E181D/Q329A mutants of PRMT7 can act on MMA and catalyze,respectively,the formation of ADMA and SDMA,while almost all the other mutants examined cannot produce either ADMA or SDMA.Moreover,while most of the mutants are inactive or have much lower activity for the production of MMA compared to that of wild-type,two of the mutants tested(I173L/F174L and Q329H)could lead to the increase of the activity.Previous experimental and theoretical studies were not able to explain the change of the product specificity and activity between the PRMT7 wild-type enzyme and mutants.Therefore,it is of considerable interest to determine the structural/energetic basis and features of interactions that allow the wild-type and the mutated enzymes of PRMT7 to direct different product formation and activity.In this study,the product specificity of PRMT7 was investigated based on the QM/MM molecular dynamics(MD)and potential of mean force(PMF)free energy simulation.The reaction state structures for wild-type and various mutants(including E172Q,E181Q,Q329A,E181D,E181D/Q329A,I173G,E172D/E181D,E181D/Q329N,I173L/F174L,Q329H and E172D)were constructed and the dynamic properties of the active sites in the reaction state were analyzed based on the results from the MD simulations.Moreover,the structural and dynamic features of the transition state and product state were studied as well based on the results from the PMF simulations.The free energy barriers were obtained for the methyl transfers in wild-type and different mutants and compared with available experimental results.The catalytic mechanism of PRMT7 and the reasons for the change of product specificity and activity as a result of mutations were clarified.The followings are the key conclusions:(1)Comparison of the results on wild-type and E172Q,E181Q and Q329A mutants demonstrated that the key factors that lead to the efficient formation of MMA in wild-type include(a)the ability of the enzyme to form the reactive and near attack conformation with the substrate Arg in the reaction complex;(b)the effective reduction of the positive charge on the guanidine group of Arg through the interactions with negatively charged E172 and E181(so Arg is able to form a more effective nucleophile for attacking the transferable methyl group);(c)strengthening the interaction at the transition states leading to transition state stabilization.The simulations also showed that PRMT7 can only produce MMA.(2)The simulations of E181D and E181D/Q329A mutants showed that they could act on MMA and add the second methyl group on the target Arg.Some important structural,electronic,and kinetic features have been identified that allow for explanation of the different product specificity characteristics and activities of wild-type,E181D and E181D/Q329A mutants.The analysis based on the formation of near attack conformation explains why E181D could only generate ADMA and E181D/Q329A could only generate SDMA.(3)The simulations also showed that both the I173L/F174L and Q329H mutants could form better near attack conformation with the substrate compared to that in wild-type and have strong salt bridge interactions with the guanidine group of Arg through E172 and E181 for the effective reduction of the positive charge.The strengthening of the interactions at the transition state are also apparent from the structures obtained from the simulations.These observations explain as to why these two mutants can be more active in the MMA production than wild type.By contrast,all the other mutants studied show the lack of the formation of good near attack conformation and/or effective way of the positive charge reduction on the guanidine group of Arg.Thus,these mutants are expected to have much lower activity for the production of MMA compared to that of wild type as observed experimentally.In summary,our studies have led to important understanding of the product specificity and activity of PRMT7 and provided theoretical basis for further investigation of the biological behavior and function of other PRMTs.The results may also be used in the future for drug discovery.