Studies Of Key Residues That Affect Phosphorylation Process Of Bacterial Response Regulator RR468

Two component signal transduction system(TCS)is the predominant signal transduction in bacteria.Due to its absence in humans and other mammals,TCS proteins are considered as potential targets for developing new antibiotics.Response regulator proteins,the second component in two-component signal transduction systems,are essential in transferring the signal delivered from the sensor histidine kinase so as to elicit an adaptive response.The phosphorylation and dephosphorylation of response regulator determine the orientation of signal transduction process,therefore,the phosphorylation and dephosphorylation sites of response regulator play an important role to exert its function.Response regulator RR468 exists in T.maritim.Because of its high expression level,highly resolved nuclear magnetic resonance spectra,and its reported crystal structure,it can serve as a good model to study the phosphorylation mechanism of TCS proteins.Previous research has shown that two non-conservative amino acid residues D+2 and T+2,located in loop ?3-?3 and loop ?4-?4,respectively,can affect the phosphorylation and dephosphorylation rate of response regulator.We performed site-directed mutagenesis experiments to these sites(M55 and K85)on RR468.The Native-PAGE demonstrates that these two mutants can affect the phosphorylation and dephosphorylation process.CPMG Relaxation Dispersion experiment shows that mutants M55A and K85E have little effects on the conformational exchange rate of three crucial loops,which means that the change of activity does not influence the dynamics of RR468.1H-15N HSQC chemical shift perturbation analysis indicates that M55A and K85E have a large effect on the structure of loop ?4-?4 and loop ?5-?5.The point-directed mutation of another conservative site D+3(M56)to Ala,eliminates the phosphorylation function of RR468 and produces a great impact on the dynamics properties of the protein.We solved the solution structure of M56A by using NMR techniques,and found that the rigid part of the structure of M56A matches well with that of the wide-type RR468.It turned out that M56A influenced the phosphorylation function by affecting the binding of Mg2+.A series of mutations were carried out to change side chain length of M56.The results show that the shorter of the side chain length,the weaker of the phosphorylation ability of RR468.This phenomenon occurs because M56 can form hydrophobic pocket with V8,L52 and V64,which is important for the stability of the ?3-?3 loop and the binding of Mg2+.If the side chain of M56 becomes shorter,it can not form the hydrophobic pocket,and then the phosphorylation function will be affected.According to the structural and functional studies of RR468,we found several key residues involved in the phosphorylation process,one of which is M56.M56 is necessary for Mg2+ binding and the stability of ?3-?3 loop.In addition to participating in the phosphate groups coordination,another important feature of Mg2+ is to stabilize the ?3-?3 loop before the phosphorylation reaction and it can help proteins to form effective active center,which is beneficial to the phosphorylation process.All the results provide theoretical basis and data support for the further study of the mechanism of the phosphorylation mechanism of response regulator.