DNA unbinding from the Yersinia pestis virulence master regulator visualized by temperature-resolved crystallography

Temperature is a fundamental physical parameter that can significantly influence the behavior of biological reactions. Moreover, temperature is a critical determinant for organismal survival, leading to the evolution of various biological temperature-sensing mechanisms. Some bacterial strains possess such abilities and utilize them to detect transitions from external to host environments, thereby triggering the expression of invasion promoters and virulence factors. Yersinia pestis one of them. Y. pestis is the bacillus causing plague, a disease also known to be responsible for some of the deathliest pandemics in human history, giving it the name of "black death" during medieval Europe. Y. pestis can detect host entry with RovA (Regulator of virulence A), a protein selectively binding DNA depending on the temperature of its environment. At lower temperatures, this inhibits the expression of virulence factors and invasion promotors. Upon host invasion and temperature increase to around 37 ?C, RovA loses affinity for its promoter, triggering the proliferation and virulence of the bacterium within its host. The temperature dependence associated to the promotion of virulence and invasion thus makes RovA a temperature-triggered master regulator responsible for Y. pestis pathogeny.Time-resolved X-ray crystallography at synchrotrons and X-ray Free Electron Lasers is a powerful method to resolve dynamic structural changes of proteins with up to femtosecond temporal and atomic spatial resolution. Here, we propose to employ this cutting-edge technology to capture a temperature- and time-resolved structural movie of the conformational changes in RovA that allow it to unbind from its promoter. This will provide unique molecular insights into the dynamics and mechanisms of how this thermal sensor controls one of the deadliest bacteria known to man.A high-resolution structure of the RovA apoprotein and of RovA bound to its promoter is already available in the Protein Data Bank (PDB). This indicates that purification and crystallization of the protein, a recurring bottleneck in crystallographic studies, is feasible. Our group has developed a setup to control the sample temperature during time-resolved serial crystallography experiments at synchrotrons and X-ray Free Electron Lasers. With an additional infrared laser to allow the increase the temperature precisely at the X-ray interaction region, we will enable the triggering of thermally dependent reactions. We anticipate that our project will reveal unique insights into the temperature-related dynamics of the master regulator responsible for Y. pestis virulence. Antibiotics are among the most potent medications against diseases that have plagued humanity throughout history, however the development of multi-resistant bacterial strains is a widely acknowledged problem. Uncovering the biological mechanisms underlying temperature triggered responses that allow bacteria to invade their host may open new avenues for medical intervention.