Molecular mechanisms of hybrid proteins in bacterial signal transduction

All living organisms need to sense and respond to the environment in order to survive. Bacteria do so predominantly via two-component systems involving sensory histidine kinase and output response regulator proteins. Upon stimulation, the kinase phosphorylates the cognate regulator, thereby regulating its effector domain activity. Via this transduction mechanism, two-component systems regulate fundamental processes such as bacterial metabolism, cell cycle, development, cell-cell communication, biofilm formation and motility, as well as virulence and pathogenesis. The regulation mechanisms of canonical histidine kinases and response regulators are understood for several signaling pathways. Therefore, recent studies have focused on special types of two-component system proteins, hybrid histidine kinases and hybrid response regulators, which combine properties of kinases and regulators into a single protein. These make up more than 20% of all histidine kinases, but both their structure and regulation mechanism remained elusive.
This thesis provides the first comprehensive characterizations of a hybrid histidine kinase, the protein ShkA from Caulobacter crescentus, which is involved in the control of stalk biogenesis, and of a hybrid response regulator, the protein LvrB from Leptospira interrogans, which is a key regulator of virulence. We employed an integrative structural biology approach, combining cryo-electron microscopy, X-ray crystallography, NMR spectroscopy and a number of biophysical and biochemical assays. In the first study, we found that ShkA constitutively adopts a closed and symmetric conformation that is catalytically inactive. Binding of the second messenger cyclic di-GMP to ShkA’s pseudoreceiver domain Rec1 causes a large conformational change that liberates the canonical Rec2 domain from the rest of the protein, leading to autokinase activity and subsequent pathway activation. We thus not only elucidated the first structure of a hybrid histidine kinase and resolved its activation mechanism, but also identified pseudoreceiver domains, whose role in the cell had remained unclear, as potential cyclic di-GMP binding modules. The work on ShkA included the development of a novel ion exchange chromatography-based procedure for the determination of enzyme kinetics. In the second study, we accomplished to resolve the structures of the hybrid response regulator LvrB in the catalytically inactive and active states, thereby unraveling its activation mechanism. In its inactive state, LvrB adopts a locked and symmetric conformation. Phosphorylation by the cognate histidine kinase LvrA, here mimicked by the phosphomimic beryllofluoride, causes a rearrangement of the N-terminal Rec domains, leading to the formation of a coiled coil next to the histidine kinase core and resulting in liberation of the nucleotide-binding domains and successive autophosphorylation.