Structural And Functional Study Of Pathogenic Mutants Of Human Adenylate Kinase 1

Human adenylate kinase(hAK1)is widely involved in the energy metabolism and balance process in various organs of the human body.In the presence of magnesium ion,hAK1 can catalyze the reversible phosphoryl transfer reaction:ADP+ADP ? ATP+AMP.Mutations in the hAK1 protein can cause moderate to severe non-spherocytic hemolytic anemia,which is a rare inherited enzyme disease.In order to reveal the pathogenic mechanism of hAK1 mutants,we recombinantly prepared 7 clinically reported pathogenic mutants of hAK1 hemolytic anemia(Q24R,G40R,G64R,R97W,R128W,R138H,delD140),and explored their structure and function.We measured the enzymatic activity of hAK1 wild-type and mutants by using coupled enzyme reaction,and the results showed that the catalytic efficiency of each mutant reduced more than 85%,comparing with that of wild type.After comparing the 2D 1H-15N HSQC spectra of wild-type and mutant,we observed apparent change of the main-chain chemical shift in R97W and R128W,which demonstrated that their conformations have changed greatly.Therefore,we compared the three-dimensional structure and dynamic characteristics of wild type hAK1 and its mutant to disclose the structural basis and pathogenic mechanism of hAK1 mutant.Firstly,we prepared R128W protein crystals by screening and optimization of crystallization conditions and determined its three-dimensional structure.The crystal structure of R128W mutant has been resolved to a resolution of 2.2 A by X-ray diffraction,the space group was P41212,and the Rwork/Rfree value was 0.2050/0.2509.The crystal structure has been successfully deposited in the Protein data bank(PDB code:7DE3).At the same time,we also obtained the wild-type hAK1 structure through homology modeling of the susAK1 crystal structure(PDB:3ADK)which has a 95.36%homology with hAK1.By comparing the three-dimensional structure of wild hAK1 and R128W mutant,we can see that the LID domain of R128W mutant which is used to bind substrate presents a more "open" state.This conformational change leads to the destruction of the interaction between residues R128,R132,R149 and the substrate,and the positive charge surface of the substrate binding pocket has also been dispersed.The above alterations may well be contributing to its substrate-binding ability and further affect its catalytic function.The results of ITC experiments showed that the affinity of R128W and R97W to the substrate decreased by 61%and 56%,respectively.Secondly,we completed the main chain chemical shift assignment of wild-type and mutant R128W,and the completion ratio of wild-type was 94.1%(177/194),and the degree of completion of R128W was 88.8%(167/194).Then we compared the dynamic characteristics of wild-type hAKland R128W mutant.The longitudinal relaxation rate R1,transverse relaxation rate R2 and heteronuclear steady-state NOE data were analyzed by sparky software.We further used Fast-Modelfree software to calculate the generalized order parameter S2 of internal motion in the time scale of ps-ns,internal motion related time τe,and conformational exchange rate Rex.The results show that the asymmetry of the mutant protein is increased,and the flexibility of the LID domain in R128W mutant is enhanced,which is consistent with the crystal structure.Additonally,the Rex value was needed to describe the dynamics models of fragment 115-120aa upon R128W mutation,indicating that this region has conformational exchange which probably caused by the conformational change of LID domain.Therefore,we speculate the fragment of 115-120aa maybe also correlated with the pathogenic mechanism of R128W mutant.In summary,we determined the structural and dynamic characteristics of R128W mutant,and revealed the structural basis of R128W mutant causing hemolytic anemia.Our results will be helpful to understand the pathogenic mechanism of R128W mutant and provides a certain theoretical basis for developing more specific and effective drug against hemolytic anemia.