Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS

<div><p>Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent <i>Bordetella pertussis</i>, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic <u>V</u>enus <u>f</u>ly<u>t</u>rap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in <i>Bordetella</i>. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.</p></div

Characterization of the VFT domains.

<p>A. Surface and cartoon representation of BvgS showing that VFT1-B is open and VFT2-A is in an apo-closed conformation. B. Ribbon representation of the open VFT1 and closed VFT2 domains. The lobes are delimited in light green and the cavities in light red. The opening angles for the VFTs are given. The linker (H9) joining VFT1 and VFT2 and the Ct loop that follows VFT2 have been included in the representation of the VFT1 and VFT2 domains, respectively. N and C indicate the N and C termini of each protomer (in A) or VFT domain (in B).</p

General organization of the BvgS periplasmic domain.

<p>A. Schematic representation of the homodimeric BvgS periplasmic portion. The protomers A and B are shown in shades of green and blue, respectively. One protomer consists of two VFT domains and a C-terminal H19 α helix. B. Ribbon representation of the X-ray structure of the BvgS periplasmic domain, the same color code as in (A) is used to show the different VFTs. The two-fold symmetry axis is indicated. C. Surface representation of the periplasmic domain of BvgS. On the left, the view angle is similar to (B), while on the right, a 90° clockwise rotation along the x-axis was applied. N and C denote the N and C termini of the two protomers.</p

BvgS heterodimers.

<p>A. Schematic representation of the BvgS heterodimers. The dimerisation/Histidine phosphotransfer domain (DHp) and the catalytic ATP-binding domain (CA) of the kinase are represented separately to show the phosphorylation cascade (arrows). B. Kinase activity levels as determined using the <i>ptx-lacZ</i> reporter for <i>B</i>. <i>pertussis</i> harboring the indicated BvgS variants and grown in standard or modulation conditions. The first panel shows the activities of the various strains. The first two express inactive homodimers, and the last four express heterodimers in which one protomer harbors a wt periplasmic portion combined with the D₁₀₂₃N substitution and the other protomer harbors the indicated periplasmic substitution(s) combined with the H₁₁₇₂Q substitution. The last three panels show the β-gal activities of the strains expressing the indicated heterodimers, with the standard errors of the mean calculated from three distinct experiments. Nicotinate (nic) and TCEP were added at the indicated concentrations (in mM). nd, no activity detected.</p

Interfaces between the VFT domains important for the kinase-on state.

<p>A. Surface representation of protomer B (in blue); the residues interacting with protomer A are shown in orange. To help visualizing these interactions, a “ghost” protomer A is represented in transparent white on top of protomer B. B. Illustration of the VFT1-VFT2 inter-protomer interface. A side view of BvgS is shown in surface representation, with the VFT1 of one protomer in green and the VFT2 of the other protomer in pale blue. A zoom delimited by a dashed orange box shows specific residues that are critical for BvgS function, as shown by mutagenesis. The side chains of Tyr₈₁ and Glu₈₆ of the β hairpin in VFT1_L1 form hydrogen bonds with Phe₃₈₆ and Arg₃₈₈ at one extremity of the VFT2 hinge, and with residues of the α helix H17. Glu₂₀₀ belongs to VFT1_L2, and its side chain makes hydrogen bonds with Asn₃₉₃ and Gly₃₉₄ at the other extremity of the VFT2 hinge. C. Illustration of the VFT2-Ct domain inter-protomer interface. In the upper panel, BvgS is shown in surface representation, with protomer A in green and protomer B in blue. A zoom shows specific residues involved in critical interactions for BvgS kinase activity. Thus, Trp₅₃₅ from H19 stacks in a hydrophobic and aromatic pocket mainly lined with VFT2_L2 residues of the other protomer, and Arg₄₇₂ and Tyr₄₇₃ from helix H16 in VFT2_L2 interact with Ser₅₂₈ and Asp₅₃₁ in the Ct loop of the other protomer. Hydrogen-bond distances are reported in angstroms.</p

<i>In vivo</i> effects of the substitutions in BvgS.

<p>A <i>lacZ</i> reporter gene under the control of the Bvg-regulated <i>ptx</i> promoter was used for determination of BvgS kinase activity in standard or modulated culture conditions. Blue and pink bars indicate kinase activity levels of bacteria producing the indicated BvgS variants and grown without or with 8 mM nicotinate, respectively, with the standard errors of the mean calculated from three distinct experiments. The middle column indicates the interfaces in which the targeted interactions are located, with inter- and intra-protomer interfaces designated ‘inter’ and ‘intra’, respectively. Nd, no β-gal activity detected; a, wild type activity and/or modulation recovered when cells were grown in the presence of TCEP; b, BvgS variants only responsive to high nicotinate concentrations (20 mM). The full set of data is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004700#ppat.1004700.s007" target="_blank">S5 Fig</a>.</p

Function of BvgS and selected homologs.

<p>A. Schematic representation of virulence regulation by BvgAS in <i>B</i>. <i>pertussis</i>. Only the virulent (Bvg⁺) and avirulent (Bvg^-) phases of the bacterium are represented for simplicity. Conditions that turn the bacteria to the avirulent phase include low temperatures and negative modulators such as sulfate or nicotinate (NA) ions. The <i>vags</i> (virulence-activated genes) are trans-activated by phosphorylated BvgA, while the <i>vrgs</i> (virulence-repressed genes) are upregulated in the avirulent phase. An intermediate phase occurs at low modulator concentrations (see text). From N to C terminus, 135 kDa-BvgS is composed of two periplasmic VFT domains, a transmembrane segment, a PAS domain, followed by a histidine-kinase (HK), a receiver (R) and a Histidine phosphotransfer (Hpt) domains that make up a phosphorelay (represented by arrows). BvgA is composed of a receiver domain and a helix-turn-helix DNA-binding domain (HTH). B. Structural organization of selected BvgS homologs, with the same color code as for BvgS. Note that the domain composition varies in the family. The cellular functions regulated by these sensor-kinases are also indicated.</p