Collections > Electronic Theses and Dissertations > Autophosphorylation of the Response Regulator PhoB
pdf

Two-component signal transduction systems are prevalent signal transduction systems in microorganisms. The essential elements of two-component systems are the sensor kinase and the response regulator. Response regulators typically receive a phosphoryl group in vivo from a sensor kinase, leading to a conformational change in the response regulator and a downstream effect. However, response regulators can also catalyze their own autophosphorylation with small-molecule phosphodonors. Using autophosphorylation to probe interactions between response regulators and small molecules might give insights potentially applicable to development of small molecule antibiotics. Prior to the work presented here, CheY was the only response regulator for which extensively characterized autophosphorylation kinetics had been published. The relationship between accumulation of phosphorylated CheY and small-molecule phosphodonor concentration remains linear up to the highest concentrations of phosphodonor tested, indicating very weak substrate binding. Here we describe extensive kinetic characterization of autophosphorylation of the Escherichia coli response regulator PhoB. Autophosphorylation kinetics differed greatly between PhoB and CheY. Specifically, the apparent rate constant for accumulation of PhoB-P appeared sigmoidal (Hill coefficient ~2) with respect to small-molecule phosphodonor concentration and to approach saturation. The data are consistent with a model in which PhoB-P forms a heterodimer with an unphosphorylated PhoB monomer, which then autophosphorylates at an enhanced rate. Potential physiological implications of PhoB heterodimers are discussed. Substitutions of nonconserved residues in the PhoB active site appeared to change the autophosphorylation reaction rate but not substrate binding. Further investigation of the link between PhoB dimerization and autophosphorylation using biolayer interferometry is proposed.