Structural And Phosphotransfer Mechanism Studies Of Protein Complex In Two-Component Signal Transduction System

Two-component signal transduction system (TCS) is the predominant signaling transduction system existing in bacteria, controlling most of the vital life regulation processes after sensing the environmental stimulus. It is an ideal drug target, due to their essential roles in the life cycle of bacteria. A conventional TCS contains two proteins, histidine kinase (HK) and response regulator (RR), which are connected by phosphotransfer. Response regulators regulate the expression of some vital genes after activated by phosphorylation. Unlike the separating ways of signaling in eukaryotes, most of histidine kinases in TCS are bifunctional, which means they can phosphorylate and also dephosphorylate the cognate response regulator under certain conditions. The phosphatase function of histidine kinase is very important for the signal base line controlling in case of cross-talk, and makes the cell regulations carry out orderly. However, the mechanism of dephosphorylation in TCS has not been clearly understood, and remains to be elucidated. It is mysterious and important to know how the histidine kinase avoids some inefficient phosphate transfer during signal transduction, due to the bifunctional property.To address these questions, this thesis systemically investigated the structures and mechanisms of the proteins HK853 and RR468 from Thermotoga maritima, and also their complexes by combining Nuclear Magnetic Resonance method and X-ray Crytallography.It was very hard to trace and investigate the phosphotransfer process during signal transduction, due to the short lifetime of phosphorylated response regulator. The phosphoryl analogue BeF3' was used as a probe to mimic phosphate group and to investigate the mechanical study of signal transduction in this thesis. The complicated exchange network, the distribution law and the kinetics properties of beryllium fluorides have been elucidated by 19F NMR. An ion pair model has been proposed to account for the impact of magnesium cation on exchange rates among beryllium fluorides.By using the phosphoryl analogue BeF3" and isotope labelling, we are able to investigate the response regulator RR468 in its unphosphorylated and phosphorylated forms. The structural characteristics of RR468 in solution display a classical ?5-?5 sandwich-like conformation.15N relaxation measurement was used to detect the dynamics of RR468 at picosecond to nanosecond time scale (ps-ns). In addition, the allosteric conformational exchanges of apo and holo RR468 at microsecond to millisecond time scale (?s-ms) were detected by CPMG relaxation dispersion experiments. We concluded that the dynamics of response regulators are relevant to their biological functions. The internal motion in fast time scale facilitates the formation of non-native hydrogen bond then decrease the activation energy barrier, while the allosteric dynamics may contribute to the catalysis for phosphotransfer and stabilize the new conformation.By using the phosphoryl analogue BeF3-, we were able to make stable samples of phosphorylated RR468 and visualize the interaction network in active site of RR468 by 19F NMR. The results indicated that the BeF3- binding network was formed by very weak chemical bonds with the sidechains of the protein.The detailed mechanism of P-RR468 dephosphorylation catalyzed by HK853 in HisKA was elucidated by 19F NMR with BeF3--RR468 as substrate. The HxxxT motif in DHp domain was found to be the most important residues participating in the dephosphorylation process. HK853 and BeF3--RR468 were successfully co-crystallized as a complex, and displayed diffraction resolution at 2.9A by using X-ray crystallography. The crystal structure of protein complex captured an intermediate conformation of HK853-BeF3--RR468 at a protecting state to avoid inefficient phosphoryl release. In the crystal structure and the highly consistent NMR results, we found that the sidechain of H260 residue is sampling two different conformations which can be regulated by pH, and the residues in CA domain participate in the interaction with P-RR through hydrophobic interaction to avoid water attack and phosphate release. Based on the mechanisms we elucidated in this thesis, we were able to design different HK mutants showing different phosphatase activities.In summary, the dynamics of response regulator RR468 was investigated by NMR at different time scales. And the component distribution, kinetics properties and magnesium impacts of beryllium fluorides have been systemically analyzed by 19F NMR. By using the BeF3" probe, the intermediate states of HK853 with its cognate response regulator RR468 have been successfully captured. High resolution crystal structure of HK853-BeF3"-RR468 complex has been determined. The structure accounts for the protecting pathway of P-RR products during signal transduction in order to avoid inefficient phosphoryl release. In this thesis, we try to explore the relationships between structure, dynamics and function of macromolecules. All the results give us a better understanding on signal transduction mechanism in two-component system. It would shed light on new anti-bacteria drug design. The new experimental methods established in this thesis can be used for other similar protein systems, and would help to elucidate more vital life mechanisms in the future.