Signal Transduction by BvgS Sensor Kinase: BINDING OF MODULATOR NICOTINATE AFFECTS THE CONFORMATION AND DYNAMICS OF THE ENTIRE PERIPLASMIC MOIETY

Abstract

The two-component sensory transduction system BvgAS controls the virulence regulon of the whooping-cough agent Bordetella pertussis. The periplasmic moiety of the homodimeric sensor kinase BvgS is composed of four bilobed Venus flytrap (VFT) perception domains followed by α helices that extend into the cytoplasmic membrane. In the virulent phase, the default state of B. pertussis, the cytoplasmic enzymatic moiety of BvgS acts as kinase by autophosphorylating and transferring the phosphoryl group to the response regulator BvgA. Under laboratory conditions, BvgS shifts to phosphatase activity in response to modulators, notably nicotinate ions. Here we characterized the effects of nicotinate and related modulators on the BvgS periplasmic moiety by using site-directed mutagenesis and in silico and biophysical approaches. Modulators bind with low affinity to BvgS in the VFT2 cavity. Electron paramagnetic resonance shows that their binding globally affects the conformation and dynamics of the periplasmic moiety. Specific amino acid substitutions designed to slacken interactions within and between the VFT lobes prevent BvgS from responding to nicotinate, showing that BvgS shifts from kinase to phosphatase activity in response to this modulator via a tense transition state that involves a large periplasmic structural block. We propose that this transition enables the transmembrane helices to adopt a distinct conformation that sets the cytoplasmic enzymatic moiety in the phosphatase mode. The bona fide, in vivo VFT ligands that remain to be identified are likely to trigger similar effects on the transmembrane and cytoplasmic moieties. This mechanism may be relevant to the other VFT-containing sensor kinases homologous to BvgS.

Keywords: Bordetella virulence; Gram-negative bacteria; Venus flytrap domain; bacterial signal transduction; electron paramagnetic resonance (EPR); membrane protein; sensor kinase; signal transduction; two-component; virulence factor.

FIGURE 1.

The sensor-kinase BvgS. a, schematic of the BvgS dimer. From the N to the C terminus, each monomer is composed of two periplasmic VFT domains, a transmembrane segment, and a PAS domain, followed by histidine kinase (HisKA), receiver (RR), and histidine phosphotransfer domains that make up a phosphorelay (arrows). BvgA is composed of a receiver domain and a DNA-binding (helix turn helix, HTH) domain and also forms dimers. b, schematic of the periplasmic moiety of BvgS. One monomer is colored green and the second one gray. The positions of the substitutions described in this work that prevent modulation by nicotinic acid are shown as red spheres centered on the Cα of each residue. The positions of previously described substitutions that similarly cause an unresponsive phenotype (21) are shown as yellow spheres (D160A, F230A, N231A, S287A, and R526A). Positions of the substitutions in the VFT2 cavity that are used in this work are shown as blue spheres. The substitutions are shown only for the green monomer.

FIGURE 2.

Nicotinate triggers fast dephosphorylation of BvgA in vivo. a, 8 mm nicotinic acid was added to B. pertussis cultures at time 0 (T0). Aliquots taken at the indicated times (in minutes and hours) were analyzed by electrophoresis in Phos-tag gels and immunoblotting using antibodies raised against BvgA. b, spontaneous dephosphorylation of BvgA. A lysate of B. pertussis grown under Bvg⁺ conditions was incubated for the indicated times at 37 °C. Aliquots were subjected to Phos-tag analysis as in a. c and d, the dephosphorylation of BvgA was performed as in a after the addition of chloronicotinate to 1 mm (c) or of the non-modulating analog nicotinamide to 10 mm (d). The numbers below the panels show the proportions of phosphorylated BvgA present in each lane relative to the amount present at time 0.

FIGURE 3.

Characterization of in vitro binding of modulators to BvgS. a, chloronicotinate binding to BvgS-p_F317A using isothermal titration calorimetry. The ITC trace and the binding curve that fit the data using a single binding site per monomer model are shown. b and c, binding curves of chloronicotinate and nicotinate, respectively, to BvgS-p_F317A as determined by thermophoresis. A representative curve is shown in both cases, and the K_d values were calculated from three separate measurements for each modulator.

FIGURE 4.

Two distinct disulfide bonds across the VFT2 cavity have opposite effects on the response to nicotinate. a, two S-S bonds were introduced across the interlobe cavity. The Cys³¹⁶–Cys⁴⁴⁵ S-S bond (red) increases BvgS sensitivity to nicotinate, whereas the Cys³⁵⁵–Cys⁴⁴² bond (blue) has the opposite effect (see Fig. 5). b, immunoblot analyses showing the BvgS variants in membrane extracts of B. pertussis. The samples were not reduced prior to electrophoresis, and, therefore, the S-S bond variants migrate slightly faster than WT BvgS (left). β-mercaptoethanol was added to the samples (right). c, distributions of the distances between Leu³¹⁶ and Ser⁴⁴⁵ and between Thr³⁵⁵ and Asp⁴⁴² over the course of molecular dynamics simulations of VFT2 (PDB code 3MPK), alone (top, apo) or with nicotinate in the interlobe cavity (bottom, holo). Histograms represent the fractional occupancy of the distances between the α carbons of Leu³¹⁶ and Ser⁴⁴⁵ (red) and of Thr³⁵⁵ and Asp⁴⁴² (black).

FIGURE 5.

β-galactosidase activities of the recombinant strains harboring BvgS variants with substitutions in the periplasmic domains. The ptx-lacZ reporter was used to determine the basal activity and response to nicotinate. The data for the recombinant strains harboring BvgS_C355-C442 or BvgS_Q463A are from Ref. . The concentrations of nicotinate in the growth medium are given in micromolar. The reducing agent tris(2-carboxyethyl)phosphine was added to the cultures at 10 mm when indicated. Note that tris(2-carboxyethyl)phosphine has no effect on WT BvgS (21). The measurements were performed at least three times, and the mean ± S.E. are shown. They are expressed in arbitrary enzymatic units, calculated as described in Ref. .

FIGURE 6.

Nicotinate affects the dynamics of the second VFT1 lobe in vitro, as shown by CWEPR. a, left panel, the model indicates the positions of the spin labels, called R1, in BvgS-p. One monomer is shown in blue and the other in gray. The spheres (yellow for 113-R1, red for 177-R1, and orange for 316-R1) are centered on the C α of the respective residues in one monomer. Center panel, the normalized spectra of BvgS-p variants alone (black) or in the presence of 20 mm nicotinate (red) are shown, with the position of the spin probe indicated above. The magnetic field is in gauss. The inset presents an enlargement of the first part of the spectrum (boxed region). The arrowheads and asterisks indicate the slow and fast components, respectively. Right panel, the schematic represents the modification of Bvg-p after nicotinate (NA) perception. The increase of the slow component in the BvgS-p_177-R1 spectrum suggests the strengthening (convergent arrows) of the VFT1_L2-VFT2_L1 interface (oval). The resulting compact block formed by the VFT2s and the VFT1_L2s affects the H19 helices so that the cytoplasmic moiety shifts to the phosphatase mode (P^ase). b, the spectra in a were simulated and are shown in stippled gray lines on top of the recorded spectra in full black lines. The correlation times (nanoseconds) of the spectral components and the occupancy of each state (percent) are indicated. c, spectra of BvgS-p_177R1 with 1, 5, 10, or 20 mm nicotinate. The effect of nicotinate is detected at the same concentrations as in vivo. d, effects of various compounds on the spectrum of BvgS-p_177R1, all used at 20 mm. Chloronicotinate and isonicotinate are in vivo modulators, unlike nicotinamide and succinate.

FIGURE 7.

Specific substitutions that make BvgS unresponsive to nicotinate affect BvgS-p dynamics in vitro. a, the CWEPR spectra (black) of WT BvgS-p_177-R1 or BvgS-p_316-R1 are superimposed onto those (red) of the indicated variants labeled at the same positions. See Fig. 6 for details. The schematics on the right show the positions of the substitutions (brown dots) and provide interpretations of their effects (shown only for one side of the dimer). Lobes colored in paler shades of blue or gray are slackened by the substitutions relative to the WT protein. First row, the F375E/Q461E substitutions at the surface of the cavity hinder nicotinate binding (crossed arrow between nicotinate (NA) and BvgS). Second row, the Q463A substitution slackens the VFT2s. The looser VFT2-VFT1_L2 interface that presumably results from slacker VFT2s is represented by divergent arrows. Third row, the small mobility decrease of VFT1_L2 is most likely caused by the W525A substitution loosening the VFT2_L1 away from the center of the dimer, therefore enabling its closer contacts with VFT1_L2 (convergent arrows). Fourth row, the D175A/Y178A substitutions in VFT1_L2 slightly increase the mobility of VFT2_L1, likely because of looser interlobe contacts (divergent arrows). b, the effect of nicotinate on the CWEPR spectra of the BvgS-p alone (black) or in the presence of 20 mm nicotinate (red). Second row, the Q463A substitution slackens the VFT2s (see a), and, consequently, nicotinate addition does not affect the BvgS-p_177-R1 spectrum, unlike for the WT protein. Third row, similarly, the slackened VFT2s_W525A prevents the effect of nicotinate on BvgS-p_177-RI. Fourth row, the D175A/Y178A substitutions in VFT1_L2 make the VFT2s slacker (see a), and nicotinate does not rigidify them. c, spectra of interest were simulated as described in Fig. 6.