Abstract
Response regulator proteins within two-component signal transduction systems are activated by phosphorylation and can catalyze their own covalent phosphorylation using small molecule phosphodonors. To date, comprehensive kinetic characterization of response regulator autophosphorylation is limited to CheY, which follows a simple model of phosphodonor binding followed by phosphorylation. We characterized autophosphorylation of the response regulator PhoB, known to dimerize upon phosphorylation. In contrast to CheY, PhoB time traces exhibited an initial lag phase and gave apparent pseudo-first order rate constants that increased with protein concentration. Furthermore, plots of the apparent autophosphorylation rate constant versus phosphodonor concentration were sigmoidal, as were PhoB binding isotherms for the phosphoryl group analog BeF3(-). Successful mathematical modeling of the kinetic data necessitated inclusion of the formation of a PhoB heterodimer (one phosphorylated and one unphosphorylated monomer) with an enhanced rate of phosphorylation. Specifically, dimerization constants for the PhoB heterodimer and homodimer (two phosphorylated monomers) were similar, but the rate constant for heterodimer phosphorylation was ~10-fold higher than for the monomer. In a test of the model, disruption of the known PhoB(N) dimerization interface by mutation led to markedly slower and noncooperative autophosphorylation kinetics. Furthermore, phosphotransfer from the sensor kinase PhoR was enhanced by dimer formation. Phosphorylation-mediated dimerization allows many response regulators to bind to tandem DNA-binding sites and regulate transcription. Our data challenge the notion that response regulator dimers primarily form between two phosphorylated monomers and raise the possibility that response regulator heterodimers containing one phosphoryl group may participate in gene regulation.
Highlights
Response regulator proteins within two-component signaling systems can autophosphorylate with small molecule phosphodonors
We propose that a heterodimer formed between a phosphorylated monomer and an unphosphorylated monomer of PhoB plays a role in PhoB autophosphorylation kinetics
Numerous lines of evidence supported the notion that association of PhoB monomers to form heterodimers affected autophosphorylation kinetics, including the protein concentration dependence of the normalized autophosphorylation time traces (Fig. 2D), the systematic deviation of the autophosphorylation time traces from a one-phase exponential (Fig. 2B), and the sigmoidicity of the plot of kapp versus PAM concentration (Fig. 3D)
Summary
Response regulator proteins within two-component signaling systems can autophosphorylate with small molecule phosphodonors. Plots of the apparent autophosphorylation rate constant versus phosphodonor concentration were sigmoidal, as were PhoB binding isotherms for the phosphoryl group analog BeF3؊. The receiver domain (Fig. 1) has a conserved (␣) tertiary structure and contains the dimerization interface for many response regulators as well as the conserved active site that catalyzes both phosphorylation and dephosphorylation [5, 10]. PhoB dimerizes through its receiver domain, allowing two output domains to come together and bind to a DNA-binding site on the promoter to regulate transcription [17]. Analysis of structures of numerous receiver domains shows that phosphorylation or binding of a phosphoryl group analog to the active site Asp leads to conformational rearrangements on the ␣4-5-␣5 surface [3, 5, 22]. We propose that a heterodimer formed between a phosphorylated monomer and an unphosphorylated monomer of PhoB plays a role in PhoB autophosphorylation kinetics
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