Function And Inhibitor Mechanism Study Of Histidine Kinase HK853 In Two-Component Signal Transduction System

Two-component signal transduction system(TCS)has mainly been found in bacteria,which is used to receive environmental stimulations then response to the signals.Totally 200-300 different TCSs were hitherto found and each kind of bacteria has about 20 TCSs.TCS proteins have similar structures and relatively conservative functions,which were significant for bacterial growth,breeding and drug resistance.Meanwhile,TCSs have not been identified in mammals including humans.Thus,TCSs are regarded as a novel antibacterial drug target.A common TCS includes two proteins,one is Histidine Kinase(HK)and the other is Response Regulator(RR).Phosphate transfer is the basic approach for TCSs to perform biological functions.In detail,HK could receive extra cellular signalstimulus and react with ATP to be autophosphorylated.After autophosphorylation,HK-P would transfer the phosphate group to RR.Phosphorylated RR(RR-P)could bind to downstream genes and regulate their expressions.HK can function as both phosphokinase and phosphatase,which could phosphorylate RR and dephosphorylate RR-P.Based on these two functions,HK is able to accurately control the relative content of RR-P to precisely regulate gene expressions.As a multifunctional enzyme,HK is often chosen as the target for TCS inhibitor sresearch.However,due to the complicated biological function and unavailable transmembrane structure of HK,detailed mechanisms of phosphate transfer and drug inhibition have not been declared yet.Herein,NMR spectrometer was chosen as major analytical instruments,a 31P NMR analytical method was established to observe catalyses of HK853-mutual transformation of ATP,ADP and AMP.We firstly finished the assignment of phosphorus-containing compounds(ATP,ADP,AMP and Pi)in the reaction.After assignment,we constantly collected the 31P NMR spectra to observe the transformation rate catalyzed by HK853.We also reported the backbone and part of the side chain assignments of HK853CA(15N-1H HSQC spectrum),which could help us to concretely analyze the changes of amino acid residues and protein conformation.According to 31P NMR experiments,we verified the inhibiting effect of Lut to HK853cp.ITC was adopted to measure the binding affinity of Lut and HK853cp.By 15N-1H HSQC spectra of titration experiments(Lut and ADP respectively titrated to HK853CA),we analyzed the different conformational changes resulted from the binding effects of Lut and ADP and further discussed the inhibition mechanism of Lut.The detailed binding sites,binding forces,and spatial position were obtained by molecular docking.All the results suggested that Lut binds to HK853CA)by 10 hydrogen bonds and a π-π stacking interaction.The binding of Lut occupies the same spatial position as ADP,and inhibits the autophosphorylation activity of HK853.Meanwhile,protein conformation and dynamic were also perturbed by the binding.Based on the above reasons,Lut could inhibit the autophosphorylation activity of HK to a certain extent.And antibacterial tests also suggested that Lut had bacteriostatic function.In this dissertation,the influences of ATP Lid and residue Y437 of HK853 on the autophosphorylation activity have also been investigated using 31p NMR and ITC experiments.The results indicated that ATP Lid and the electric charge of residue 437 were key factors to autophosphorylation activity.In the last part of this dissertation,a new enzyme activity of HKs has been found,which is HK can transform ADP to ATP and AMP(ATP synthesis activity).’ P and 15N-1H HSQC NMR experiments were recorded to study the critical regions and residues of HK853 to this ATP synthase activity preliminarily.We also speculated the mechanism of this enzymatic reaction.The results suggested that R430,N376 and K383 of HK853 were critical residues for ATP synthase activity.The dimerization and histidine phosphotransfer domain(DHp domain)and ATP Lid of CA domain also participated in regulating this reaction.Mutation of N380A which significantly decrease the binding affinity of ATP and HK853 could accelerate the reaction.The phosphate group reception residue H260 showed no obvious effect on the HK853’s ATP synthase activity.In the absence of ATP,HK853 could transform part of ADP to ATP and AMP in a very fast reaction rate.And RR468 could be phosphorylated by the phosphate group originally came from ADP.Therefore,we inferred that this ATP synthase activity might play a crucial role under extreme environment.And we also speculated that the reaction formula is 2ADP→ATP+AMP,which is one ADP breaks a high-energy phosphate bond,releases energy and a phosphate group then transfers to AMP,the other ADP forms a high-energy phosphate bond,gets energy and a phosphate group then transfers to ATP.In summary,we carefully studied the autophosphorylation inhibition mechanism of Lut to HK853.The autophosphorylation and ATP synthase mechanisms of HK853 were also preliminarily studied.These results provide experimental basis for research and development of HK inhibitor.And our study also provides a profound foundation for HK’s enzymatic activities especially ATP synthase activity.