Structural And Dynamic Studies On Response Regulator PhoB_N In Two-Component Signal Transduction System

Two-component signal transduction system?TCS?is most important cell signaling systems which control many bacterial adaptive behaviors and pathways.This system consists of two different proteins,a histidine kinase?HK?which is a transmembrane sensing protein,and a response regulator?RR?which is located in the cytoplasm.Histidine kinase usually reacts with signals in an extracellular environment.Communication between proteins occurs via phosphoryl group transfer from a histidine of HK to an aspartate of the RR.The phosphorylated response regulator protein acts as a repressor or activator and modulates the expression of downstream genes.The transduction processes of TCS system are different,mainly reflected in the many functions of HK protein,which can accomplish both self-phosphorylation and phosphorylation or dephosphorylation of RR protein.Although the signal transduction pathway of TCS is clear,the individualized molecular mechanism of its signal regulation remains unclear.The phosphorylation rate and conformational change of different RR proteins have their own characteristics,so it is of great biological significance to study the structure and phosphorylation process of RR protein.The response regulator protein PhoB from PhoR/PhoB system has been studied.It mediates the response to changing concentrations of the environmental phosphate Pi signal.PhoB has two domains,one is receiver domain?PhoB_N?which has phosphorylated site and the other is effector domain which can bind with DNA.The crystal structures of active and inactive PhoB_N have been solved by X-ray,respectively.It shows a conserved?5?5 folding,forming a homodimer with an?1-?5face in the apo form and an?4-?5-?5 dimer in the phosphorylated form.The?1-?5dimer interface is rarely observed,and the relationship between the conformational changes and the phosphorylation mechanism of PhoB_N is unclear.In this paper,the structure and dynamics properties of PhoB_N in solution have been studied by liquid nuclear magnetic resonance?NMR?technology.The secondary structure of PhoB_N was predicted by the backbone assignments,and results showed that PhoB_N is dominated by?5?5 folding that is the same as the crystal structure.Concentration dilution experiments and backbone relaxation parameters showed that PhoB_N exists in equilibrium between monomer and dimer states.Using paramagnetic relaxation enhancement experiments,we demonstrated that the dimer of apo PhoB_N forms several transient dimer interfaces in solution.This finding suggested that,in addition to the monomer-to-dimer exchange,the inactive conformation of PhoB_N has different domain arrangements which are independent of phosphorylation.BeF₃^-as a phosphoryl acid analogue,can stabilize the active state of response regulator protein and help to study the phosphorylation dynamic process of RR protein.For most of RRs,the binding of BeF₃^-is magnesium-dependent,but PhoB_N can be partially phosphorylated without Mg²⁺and can be completely phosphorylated with Mg²⁺.PhoB_N has multiple conformational changes in solution,and the NMR spectrum gives inhomogeneous signals and makes it hard to investigate.PhoB_N^F20D20D becomes a monomer in the solution and has the same phosphorylation property as the wild type PhoB_N.Therefore,the mutant PhoB_N^F20D was used to explore the phosphorylation mechanism.We found that apo PhoB_N^F20D has a pre-active conformation in solution and BeF₃^-can stabilize this conformation without Mg²⁺.CPMG and CEST experiments have confirmed that there are conformational exchanges of PhoB_N^F20D in solution.We further studied the phosphorylation mechanism of PhoB_N^F20D in the presence of Mg²⁺and found that Mg²⁺binding caused chemical shift changes in helix?4 and loop?3-?3.Residues in these regions were mutated and found that residues D+1?W54?,D+2?M55?,and T+2?R85?reduce the autophosphorylation rate and D+3?L56?completely abolish the autophosphorylation ability.W54 is close to the phosphorylation site D53,and it has two conformations after phosphorylation.In conclusion,the conformational exchanges of apo PhoB_N in solution were studied by liquid NMR spectroscopy.It was proposed that the protein exists monomer and dimer equilibrium,and there were multiple interaction modes at the dimerization interface.Through the study of PhoB_N^F20D mutants,we found that it also has a conformational exchange between free and pre-active states when protein is in a monomer,and BeF₃^-can help to stabilize its phosphorylation state.At the same time,the important influence of the binding of Mg²⁺on the protein structure and the phosphorylation process were explored.Our research provides experimental basis for exploring the signal transduction mechanism of the TCS,and it also helps to further reveal the mechanism of the phosphorylation of the response regulator proteins.