Activation of Bacterial Histidine Kinases: Insights into the Kinetics of the cis Autophosphorylation Mechanism

Two-component systems consisting of an input-sensing histidine kinase (HK) and an output-generating response regulator (RR) are one of the key apparatuses utilized by bacteria for adapting to the extracellular milieu. HK autophosphorylation is shown to occur primarily in trans (intermolecular) and more recently shown to occur in cis (intramolecular). Although the catalysis of HK activation remains universal, the reaction scheme for evaluation of the kinetic parameter differs between these designs and cis mode largely remains unexplored. We combined experimental and theoretical approach to unravel two-step mechanism of activation of three cis mode HKs of M. tuberculosis. The new mathematical model yields best-fit parameters to estimate the rates of HK-ATP association and HK autophosphorylation. ABSTRACT Two-component signaling systems (TCSs) are central to bacterial adaptation. However, the mechanisms underlying the reactions involving TCS proteins and their reaction rates are largely undetermined. Here, we employed a combined experimental and theoretical approach to elucidate the kinetics of autophosphorylation of three histidine kinases (HKs) of Mycobacterium tuberculosis, viz., MtrB, PrrB, and PhoR, all known to play a role in regulating its virulence. Using wild-type and mutant proteins, we performed dimerization assays, thermophoretic-affinity measurements, and competition-based phosphorylation assays to establish that for HK, MtrB autophosphorylation occurs in cis, similar to what has been proposed for the PhoR and PrrB HKs. Next, to determine the kinetics of cis autophosphorylation, we used a quantitative high-throughput assay and identified a two-step mechanism of HK activation, involving (i) the reversible association of HK with ATP, followed by (ii) its phosphorylation. We developed a mathematical model based on this two-step cis mechanism that captured the experimental data. Best-fit parameter values yielded estimates of the extent of HK-ATP association and the rates of HK autophosphorylation, allowing quantification of the propensity of HK autophosphorylation. Our combined experimental and theoretical approach presents a facile, scalable tool to quantify reactions involving bacterial TCS proteins, useful in antibacterial drug development strategies. IMPORTANCE Two-component systems consisting of an input-sensing histidine kinase (HK) and an output-generating response regulator (RR) are one of the key apparatuses utilized by bacteria for adapting to the extracellular milieu. HK autophosphorylation is shown to occur primarily in trans (intermolecular) and more recently shown to occur in cis (intramolecular). Although the catalysis of HK activation remains universal, the reaction scheme for evaluation of the kinetic parameter differs between these designs and cis mode largely remains unexplored. We combined experimental and theoretical approach to unravel two-step mechanism of activation of three cis mode HKs of M. tuberculosis. The new mathematical model yields best-fit parameters to estimate the rates of HK-ATP association and HK autophosphorylation.