The PH-Regulatory Mechanism Of HK853 Investigated By Selective Labeling

The two-component signal transduction system(TCS)is the main means of bacteria sensing and responding to the various extracellular stimuli.Most physiological processes of bacteria,such as perceptual osmotic pressure,metabolism of nutrients,synthesis of cell walls and cell proliferation and division,are regulated by two-component systems.Two-component signal transduction systems are widely found in bacteria,and are found in some fungi and plants,but they have not been found in humans and other mammals so far.Therefore,the two-component system is considered as a potential target for antimicrobial agents.The two-component signal transduction system consists of histidine kinase(HK)and response regulator(RR).HK can sense environmental stimulation and transmit this stimulation signal to the downstream RR through the transfer of phosphate groups,while the phosphorylated RR can regulate signal output through the interaction with gene or target protein,or the cell regulation can be terminated by dephosphorylation.In the two-component signal transduction system of bacterial,most of HK have multiple functions,which can not only achieve self-phosphorylation and transfer phosphate groups to RR protein,but also catalyze the dephosphorylation of RR.The crystal structure of the complex formed by the intracellular full-length protein of HK853(HK853P)and substrate RR468 is not the same in different pH.His260 residue is the key site of HK853 participating in the dephosphorylation reaction,and the crystal structure of the complex indicates that the spatial position of the imidazole side chain is significantly different at different pH.The imidazole of side chain of histidine is often used to flexibly regulate the reaction process through different protonated states in the catalytic reaction.However,due to the resolution of crystal structure,the protonated state of the key residue His260 is not clear during the catalytic dephosphorylation reaction.Analyzing the proton exchange process of His260 site in HK853 for kinase and phosphatase function switching is the key to study the acid-base regulation mechanism of HK.Nuclear magnetic resonance spectroscopy has advantages in studying the interaction between biomacromolecules,but because the complex formed by HK853cp and substrate RR468 has a molecular weight of more than 85 kDa,ordinary isotope labeling methods cannot obtain resolvable NMR signals.In this paper,a stable phosphorylation complex of HK853 and RR468 was obtained by using the phosphate group analog BeF3-.A selective labeling method was used for the isotopic labeling of the groups ?l-C on the side chains of the histidine residues of HK853 DHp domain,and a clear NMR signal was obtained.The different histidine sites were subjected to site-directed mutagenesis,and the 1H-13C HSQC spectrum signal was assigned.The pH titration of 1H-13C HSQC determined the pKa value of HK853 and its complex proton exchange process.The pKa value of His260,the active site,was significantly different from that of histidine at other inactive sites,and the pKa value corresponded to the optimal pH range of HK853DHp for phosphatase function obtained by dephosphorylation.In addition,a series of HK853DHp mutants were obtained by site-directed mutagenesis.The phosphatase activity of HK853DHp and the mutants were determined by dephosphorylation assay.Combined with the results of 19F NMR pH titration,the effect of Thr264,another conserved residue in the DHp domain,on the phosphatase function of HK853 was determined.In summary,the high resolution NMR spectra of HK853 and its complex HK853-BeF3--RR468 were obtained by 13C?1-His selective isotope labeling.The pKa value was determined by pH titration experiment of 1H-13C HSQC,and the protonation state of His260 site when HK853 performe phosphatase function was determined.The 19F NMR pH titration and phosphatase activity assay confirmed that the synergy of Thr264 site with His260 affects the phosphatase function of HK853,further elucidating the pH regulation mechanism of histidine kinase HK853.The research provides new data for the study of the regulation mechanism of two-component signal transduction system and helps to open up new antibacterial drug design ideas.At the same time,the use of amino acid side chain selective isotope labeling to study the molecular mechanism of TCS provides a reference for the study of simila r protein systems.