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

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.

Keywords: Mycobacterium tuberculosis; autophosphorylation; cis autophosphorylation; histidine kinase; mathematical modeling; two-component signaling.

FIG 1

Analysis of HK autophosphorylation using mutant complementation assay. (A) Autophosphorylation analysis of MtrB proteins. Wild-type (WT) MtrB or the MtrB^H305Q or MtrB^N419D mutant was tested in autophosphorylation reaction for 90 min per the protocol in Materials and Methods. The graph below the autoradiogram represents densitometric analysis of the autoradiogram (in arbitrary units [A.U.]). (B) Analysis of trans phosphorylation by competition experiment. MtrB and MtrB^H305Q proteins were coincubated in various molar ratios as indicated for 10 min. The autophosphorylation reaction was performed for 60 min. (Top) Autoradiogram; (bottom) CBB-stained gel. The graph below the autoradiogram represents densitometric analysis of the autoradiogram. (C to E) Microscale thermophoresis measurements for determination of interaction affinities of MtrB-GFP with WT MtrB (C), MtrB^H305Q (D), and MtrB^N419D (E). All graphs were best fit to means ± standard errors of the means (SEM) from three independent experiments. F_norm, normal fluorescence. (F) MtrB dimerization analysis using nonreducing SDS-PAGE and fluorescence imaging assay by coincubating 2 µM MtrB-GFP with MtrB, MtrB^H305Q, or MtrB^N419D (at a concentration of 5 µM). Imaging was done per the protocol described in Materials and Methods. Lane M contains molecular size standards.

FIG 2

Equivalence of HTA platform and PAGE/autoradiography for determining HK autophosphorylation kinetics. (A) Evolution over time of labeled MtrB measured using HTA and PAGE/autoradiography independently and normalized to the maximum measured values in the respective assays. MtrB (5 µM) and 50 µM unlabeled ATP and 50 nM labeled ATP were used for the reaction per the protocol described in Materials and Methods. The left-hand y axis shows normalized MtrB·P_H concentration obtained using HTA, and the right-hand y axis shows normalized MtrB·P_H band intensities. The evolution over time of the normalized concentrations of MtrB·P_H measured using the HTA platform was nearly identical to the normalized intensities measured using PAGE/autoradiography (P = 0.8 using two-tailed Student’s t test with paired samples and unequal variance). (B) Band intensities from PAGE/autoradiography showed linear correlation with corresponding concentrations of labeled MtrB obtained using the HTA platform (see Fig. S3 in the supplemental material). The data are shown as means ± standard deviations (SD) (n = 3).

FIG 3

Time course analysis of formation of phosphorylated HK. Amount of labeled HK and PrrB (A), PhoR (B), and MtrB (C) formed at various time points determined using the HTA platform in the presence of various ratios of unlabeled/labeled ATP used in the reaction. The amounts of unlabeled ATP employed that were used along with ~50 nM labeled ATP are indicated. HKs were used at the following concentrations in the reaction, 10 µM for PrrB and PhoR and 5 µM for MtrB. The data are shown as means ± SD (n = 3).

FIG 4

Fits of model predictions to data of PrrB autophosphorylation kinetics. Best-fits (solid lines) along with 95% confidence intervals (dashed lines) of model predictions (equation 10) to the data in Fig. 2 (symbols). Fits are obtained simultaneously to all the data obtained at various concentrations of unlabeled ATP as indicated in the panels. (A) 25 µM, (B) 50 µM, (C) 200 µM, and (D) 300 µM. (Overall R² = 0.9.) The resulting parameter estimates are listed in Table 1. The data are shown as means ± SD (n = 3).

FIG 5

Validation of HK autophosphorylation kinetics with mathematical model. Model predictions (solid lines) using parameter values estimated in Fig. 3 (Table 1) compared with data (symbols) of labeled HK as indicated, (A) PrrB, (B) PhoR, and (C) MtrB from independent experiments using the HTA platform. Concentrations of HKs used for the reaction were 5 µM for MtrB and 10 µM for PrrB and PhoR along with 50 µM unlabeled ATP and ~95 nM labeled ATP. The data are shown as means ± SD (n = 3).

FIG 6

Validation of two-step HK autophosphorylation mechanism. Time course analysis of formation of labeled MtrB^H305Q·ATP complex using the HTA platform in the presence of various concentrations of unlabeled ATP as shown in the panels. (A) 5 µM, (B) 10 µM, (C) 100 µM, (D) 150 µM, (E) 200 µM. A fixed concentration of labeled ATP (20 nM) and 5 µM mutated MtrB^H305Q was used for all reactions. Best-fits (solid lines) along with 95% confidence intervals (dashed lines) of model predictions (equation 10) are obtained by simultaneous fitting to the data using k_p = 0 (absence of phosphorylation), K_e = 1.7 mm⁻¹, and ϕ as adjustable parameter. The data are shown as means ± SD (n = 3).

FIG 7

Summarized model of findings reported in this study. Step 1 indicates the presence of rapid and reversible association of ATP with HK. Step 2 is the slow autophosphorylation of HK in response to stimulation by ligand, which is the rate-limiting step. P, phosphate group.