Periplasmic domain of the sensor-kinase BvgS reveals a new paradigm for the Venus flytrap mechanism

Abstract

Two-component sensory transduction systems control important bacterial programs. In Bordetella pertussis, expression of the virulence regulon is controlled by the unorthodox BvgAS two-component system. BvgS is the prototype of a family of sensor-kinases that harbor periplasmic domains homologous to bacterial solute-binding proteins. Although BvgAS is active under laboratory conditions, no activating signal has been identified, only negative modulators. Here we show that the second periplasmic domain of BvgS interacts with modulators and adopts a Venus flytrap (VFT) fold. X-ray crystallography reveals that the two lobes of VFT2 delimitate a ligand-binding cavity enclosing fortuitous ligands. Most substitutions of putative ligand-binding residues in the VFT2 cavity keep BvgS active, and alteration of the cavity's electrostatic potential affects responsiveness to modulation. The crystal structure of this VFT2 variant conferring constitutive kinase activity to BvgS shows a closed cavity with another nonspecific ligand. Thus, VFT2 is closed and active without a specific agonist ligand, in contrast to typical VFTs. Modulators are antagonists of VFT2 that interrupt signaling. BvgAS is active for most of the B. pertussis infectious cycle, consistent with the proposed mechanism.

Fig. 1.

X-ray structure of VFT2. (A) The overall structure of the protein is represented as a ribbon diagram, with the acetate and glycerol molecules shown as red and green sticks, respectively. Lobe 1 is at the left and the two-stranded hinge (S7 and S13) is in the middle. α-Helices and β-strands have been colored cyan and magenta, respectively. VFT2 also harbors two 3₁₀ helices, H4 and H5. (B and C) Major interactions between the molecules present in the VFT2 cavity and the lobes of the protein (B, acetates and C, glycerol). Lobes I and II are in pale green and orange, respectively, with the hinge strands shown in gray. The H bonds are shown as dotted yellow lines and the H₂O molecules as red spheres. For the sake of clarity, Van der Waals interactions are not represented.

Fig. 2.

β-gal activities of recombinant strains harboring BvgS variants with substitutions in the VFT2 cavity. A lacZ reporter placed under the control of the Bvg-regulated ptx promoter was used to assess Bvg activity. The first panel shows the activities of the various strains grown in SS medium in the absence of modulation calculated as in ref. . The effects of the negative modulators MgSO₄ (○) and nicotinic acid (■) on β-gal activity are shown in the remaining panels, where 100% represents the activity of bacteria grown without modulators.