MECA VENUS

The predominant signal transduction systems in bacteria, two-component systems are essential to enable microorganisms to adapt to changes of their environment. They regulate important developmental programs including bacterial virulence. Typically, they are composed of a transmembrane sensor-kinase protein and a cytoplasmic response regulator. Perception of a chemical or physical signal by the sensor leads to kinase activation and autophosphorylation, and then transfer of the phosphoryl group to the response regulator. Thus activated, the latter mediates a specific, frequently transcriptional, cellular response. The whooping cough agent Bordetella pertussis colonizes the upper respiratory tract of humans. Its virulence regulon is controlled by the two-component system BvgAS. At 37°C and in laboratory growth conditions, the BvgAS system is activated, leading to the transcription of the virulence regulon, including genes for B. pertussis’s adhesins and toxins. The virulent Bvg+ phase of B. pertussis is necessary for infection. Switching to the avirulent Bvg- phase is triggered by the addition of negative modulators. Thus, BvgS might be active by default and inactivated by antagonists at specific stages of the bacterium’s life cycle. BvgS is a hybrid sensor-kinase harbouring several cytoplasmic domains that mediate a complex phospho-transfer cascade. It also contains two periplasmic ‘Venus flytrap’ (VFT) domains in tandem and a cytoplasmic PAS domain before the kinase. Ubiquitous in nature, VFT domains usually function along a clamshell model, with two lobes that enclose specific ligands between them. Conformational changes of VFTs between open and closed forms upon ligand binding are widely used for transport, or also for signalling such as in the ion-channel coupled Glu receptors of higher eukaryotes. BvgS is the prototype for a large family of multi-domain sensor-kinases that harbour periplasmic VFT perception domains, which represents a new paradigm of VFT receptors involved in signal transduction. The molecular mechanisms of signal perception and transduction by these proteins remain unexplored. We have recently obtained the crystal structure of the entire periplasmic domain of BvgS, showing a novel architecture for VFT receptors. The periplasmic domain of BvgS is dimeric, with extensive interfaces between protomers. Our preliminary data have indicated that the conformations of the VFT domains determine BvgS activity and that the interprotomer interfaces are critical for function. This new structure and the tools that we have developed for the functional analysis of BvgS put us in a good position to decipher the molecular mechanisms of signalling by using an integrated approach that combines functional analyses, biochemistry, structural biology and in silico modelling. We will identify critical regions of the VFT domains that determine the active conformation of BvgS and the mechanisms by which negative signals are perceived and transmitted, using site-directed mutagenesis and a functional assay in bacterio. The conformation and dynamics of BvgS and its mutants will be investigated by crystallography and by modelling, using molecular dynamics simulations and coarse-grained approaches. Simulations will also be performed of the VFT domains with the transmembrane segment in a lipid environment. This will be combined with biochemical approaches to explore the topology and dynamics of the segment linking the periplasmic and cytoplasmic moieties, and of the linker between the PAS and kinase domains. The function and topology of the PAS domain will be characterized. Our program will enable us to propose a model that describes the molecular mechanisms of signal transduction in this family of VFT sensor-kinases. This will enlarge our knowledge on two-component-mediated signal transduction in bacteria, as well as lay the bases for new avenues of targeted therapeutic intervention.