Structural And Functional Studies On Human Adenylate Kinase 4

Aden ylate kinases(AKs) are ubiquitous enzymes which are involved in energy metabolism and homoeostasis of cellular adenine nucleotide composition.They catalyze reversible transfer ofγ-phosphate group from Mg2+ATP(or GTP) to AMP, releasing Mg2+ADP(or GDP) and ADP.AKs belong to the nucleoside monophosphate kinase(NMPK) family that includes other members such as guanylate kinases,thymidylate kinases,and UMP/CMP kinases.All of these enzymes share a common typicalα/βfold that consists of aβ-sheet CORE surrounded byα-helices,with a P-loop motif at the N-terminus that binds the phosphoryl donor. All AKs contain a central CORE domain,a LID domain,and an AMP-binding domain called NMPbind.The CORE and NMPbind domains are conserved in all AKs, whereas the LID domain is quite different.Six AK genes named AK1-AK6 have been identified in vertebrates.Adenylate kinase 4(AK4) is a unique member with no enzymatic activity in vitro in the adenylate kinase(AK) family although it shares high sequence homology with other AKs.It remains unclear what physiological function AK4 might play or why it is enzymatically inactive.Most residues within active site are conserved in human AK4 except residue 159,where an arginine conserved in other AKs is replaced with a glutamine.Structural analysis revealed that,while AK4 retains the capability of binding nucleotides,AK4 has a giutamine residue instead of a key arginine residue in the active site well conserved in other AKs.Mutation of the glutamine residue to arginine(Q159R) restored the adenylate kinase activity with GTP as substrate.In this study,we strive to establish the functional importance of AK4.When cells were exposed to hypoxia,the AK4 protein level increased significantly Moreover,we tested the AK4 levels in a commonly used transgenic mouse model of ALS,a neurodegenerative disease in which oxidative stress has been implicated,the AK4 level was significantly higher in the spinal cords of ALS mice.The results support that AK4 protein levels increase in response to different stress in cultured cells as well as in an animal model of human disease,suggesting that AK4 might provide protective benefits to cells under stress.Laterly,we showed that small hairpin RNA (shRNA)-mediated knockdown of AK4 in HEK293 cells with high levels of endogenous AK4 resulted in reduced cell proliferation and cell death.Furthermore, we found that AK4 over-expression in the neuronal cell line SH-SY5Y with low endogenous levels of AK4 protected cells from H2O2 induced cell death.These results indicate that the enzymatically inactive AK4 is a cell stress protective protein critical to cell survival and proliferation.Proteomic studies revealed that the mitochondrial ADP/ATP translocases(ANTs) interacted with AK4 and more interaction can be detected when cells were exposed to H2O2 treatment.It is likely that AK4 function by modulating its interaction with the mitochondrial inner membrane protein ANT.In vitro pull-down experiments further confirmed the protein-protein interactions between AK4 and ANT,further more,the a andβsubuinits of mitochondrial ATP synthase F1 complex are also found to be involved in the protein interactions with AK4.ANTs are important in mitochondrial ADP/ATP transport in the cell energy metabolism system,which also be reported to form into big complex(ATP sythasome) with mitochondrial ATP synthase.In other words,the proteins identified to be interacted with AK4 play key roles in process of mitochondrial ATP synthesis and translocation.Combined with the result of AK4 retains the capability of binding nucleotides,which suggests that AK4 may be also involved in the process of mitochondrial ATP synthesis and translocation through interaction with ANT and ATP synthase.It is well known that motion of LID and NMP-binding(NMP_bind) domains in adenylate kinase(AK) is important in ligand binding and catalysis.However,the nature of such domain motions is poorly characterized.One of the critical hinge regions is hingeⅣ,which connects the CORE and LID domains.In addition,the hingeⅣcontains a strictly conserved residue,L 171,in the AK family.To investigate the role of hingeⅣ,crystal structure of human adenylate kinase 4(AK4) L171P mutant was determined.This mutation dramatically changes the orientation of the LID domain,which could be described as a novel twisted-and-closed conformation in contrast to the open and closed conformations in other AKs.This mutant provides a new example of domain motions in AK family.Collectively,these results indicate that the enzymatically inactive AK4 is a cell stress protective protein critical to cell survival and proliferation and it is likely to function by modulating its interaction with the mitochondrial inner membrane protein ANT and ATP synthase.So it is possibly that AK4 involved in the process of mitochondrial ATP synthesis and translocation through interaction with ANT and ATP synthase.The crystal structures of AK4(L171P) provide a direct model of hinge regions regulate protein conformational change.