Dimerization and Conformational Exchanges of the Receiver Domain of Response Regulator PhoB from Escherichia coli

PhoB is a response regulator of PhoR/PhoB two-component system from Escherichia coli that is involved in the environmental phosphate regulation. It has been reported that the N-terminal receiver domain (PhoB_N) forms a dimer using the α1−α5 face in the apo state and a dimer using the α4−β5−α5 face in the active state investigated by X-ray crystallography. However, it is not clear whether the conformational switch of the dimer is dependent on phosphorylation. Here, we report the NMR studies of PhoB_N in solution in its apo form. We observed that the secondary structural fragments of apo PhoB_N characterized by NMR are almost the same as those determined by crystallography, but the NMR spectrum of PhoB_N shows inhomogeneous amide signals. 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. It provides an experimental data for the conformational selection mechanism of the phosphorylation of PhoB<sub>N