This research program is pioneering structure-function studies of Ser/Thr protein kinases (STPKs) that serve as essential environmental sensors in the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb causes tuberculosis (TB), and it infects one third of the world's population. TB kills ~2 million people annually, and drug resistant strains are emerging rapidly. To target the Mtb STPKs with new therapeutics, basic research is needed to define the biological functions and regulatory mechanisms of these essential receptor kinases. In the first grant period, we determined the first structures of the Mtb STPKs and established the current paradigm that dimerization and phosphorylation mediated by two interfaces activate bacterial STPKs. We also developed novel approaches to identify STPK substrates in Mtb. These studies afford the opportunity to focus a broad array of methods--including fluorescent kinase fusion proteins, biochemical assays and X-ray crystallography--to establish general principles of bacterial STPK signaling. Our revised aims will achieve three immediate milestones in two years: 1. Define the location of the master Mtb STPK PknB and key substrates to test the hypothesis that PknB forms a protein complex that regulates cell wall biosynthesis. 2. Discover networks of kinase cross-phosphorylation to test the idea that the STPKs regulate each other in specific hierarchical patterns. 3. Analyze the first crystal structure of a bacterial pseudokinase alone and in complex with a cellular regulator to test our new model that PknB regulates the oligomerization of the Mtb flippase for peptidoglycan precursors. Our work to date established the conceptual framework and the feasibility of these aims. We propose several groundbreaking studies, including spatial and temporal mapping of kinase localization in vivo. We also will test our new model for recognition of ubiquitous pThr-binding modules called FHA domains. Because of the central roles of kinase signaling in cellular physiology, the direct focus on Mtb STPKs as pharmaceutical targets, and the worldwide impact of TB, our studies will have high medical significance.
TB has re-emerged as the world's leading infectious disease, and the rapid proliferation of MDR (resistant to 2-3 antibiotics) and XDR (resistant to 5 antibiotics) TB has increased the urgency to develop new therapeutics. This project provides the underlying biological knowledge to support ongoing programs at several institutions--including Novartis (Singapore), GSK (Bangalore), Plexxikon (Berkeley), Vertex Pharmaceuticals (Cambridge), Ranbaxy Pharmaceuticals (Delhi) and the New Medicines for TB initiative (Lausanne)--to develop preclinical candidates that inhibit Mtb PknB or PknA. Our studies defining the general regulatory mechanisms of STPKs also will aid in understanding and treating many other diseases in which protein kinases play key roles.
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