Computational Investigation On Mutation Effect Of Dnmt3a R882H And Allosteric Effect Of Akt1 Phosphorylation

Acute myeloid leukemia（AML）is one of the most frequent acute leukemia in adult at present,and the molecular cause of morbidity is yet unknown.Recently deep-sequencing has been widely used for those patients,and high-frequent mutations have been observed in genome.As a result of bioinformatics analysis,defect in epigenetics and signaling transduction has been considered as etiology for blood cancer as in other cancers.Thus,investigation on gene mutation and protein structure would help to understand the molecular pathogenesis in the sense of structure-function relationship.This work focuses on DNA methyltransferase Dnmt3a and Akt1 kinase,two most important elements in epigenetics and PI3K/Akt signaling pathway.R882H mutation effect of Dnmt3a and allosteric effect of monophosphorylated Akt1 kinase were selected as case-study objects.DNMT3A R882 mutation would lead to DNA hypomethylation in patients.In vitro experiment has concluded that the R882H mutation decreased catalytic activity.However,the mutation site is far from the active site,by a distance of 20 angstroms.How to understand the distal effect?First,a high volume of pre-reaction state was collected from classical molecular dynamics simulation.Then two sample t-test was used to enrich the most significant fluctuations of residues.The detailed changes in catalytic site were compared between the wide type and mutant.Finally,the transduction pathway from distal mutation to catalytic center was located.The most important findings are 1)E667,V684,G685,E697,W698,K829,V830,I833,S881,and R885 present the highest significant fluctuation owing to the R882 mutation;2)coenzyme S-adenosyl-L-methionine present different conformation in the mutant in the rate-determining step of methyl transfer;3)secondary structure H11 helix seems to be responsible for transducing mutation effect from the 882 site to the catalytic site;4)interestingly,the mutation stabilized both the rate-determining transition state and rate-determining intermediate（pre-reaction state in this study）.In kinase study,consistent activation is believed to transport cascading signal in cancer development.There are two phosphoryl sites in Akt1 kinase,S473 and T308.Phosphorylation of T308 is essential for fully activation of Akt1 kinase.However,the molecular mechanism for the consequence was unknown.ADP-Akt1 and ATP-Akt1 complexes were taken as objects to understand activation and non-activation.It was found that,1)doubly protonated His194 maintained the hydrogen bond between His194 and p T308,which stabilized the active state of Akt1 kinase;2)ATP could maintain the salt bridge and allosteric pathway to further protect p T308from dephosphorylation;3)through the transition from inactive state to active state,G-loop could form hydrogen bond with ATP to stabilize its location in active site,meanwhile,water molecules were increased in catalytic pocket;4)catalytic mechanism by quantum chemistry calculations revealed that water in active site could coordinate with Mg²⁺to replace the coordination between Mg²⁺andγphosphate.The releasedγphosphate further perform nucleophilic attack towards substrate to finish phosphate transfer reaction.Our results casted light on the pathogenic mechanisms of Dnmt3a R882H mutation and allosteric mechanisms of active state Akt1 on molecular level.More importantly,the strategy used in this work is suitable for exploring more mutation effects in post-genomics era.