Protein-protein interactions and structural switching of the bifunctional Chlamydia trachomatis protein, Scc4

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PROJECT SUMMARY The role of the bi-functional Chlamydia trachomatis protein, Scc4 (formerly Ct663), in regulating the type III se- cretion system (T3SS) and the essential, developmental cycle (between infectious elementary bodies and ac- tively dividing, reticulate bodies) is not well-understood at the molecular level. Scc4 is a unique protein that functions (i) as a T3SS chaperone with Scc1 to regulate secretion of CopN (an essential virulence factor) and (ii) as a transcription factor regulating 66-RNA polymerase holoenzyme. Based on progress made during the last funding period, the switching mechanism proposed for Scc4’s dual functions involves a conformational change in its 3-dimensional structure. Because Scc4 has two essential functions and is unique to C. tracho- matis, it is a novel target for developing anti-virulence drugs. To develop drugs targeting the C. trachomatis T3SS and developmental cycle, hit-compounds must first be identified and their effects on Scc4’s structure, protein-protein interactions, and functions characterized. The long-term goal of the proposed research is to de- velop drugs targeting Scc4’s functions, which would prevent the bacteria from spreading, likely curing C. tra- chomatis directly and certainly mitigating the development of antibiotic-resistant strains, when combined with current therapies. The overall objectives of this application are to increase our understanding of the conforma- tional changes and binding sites that regulate Scc4’s switching mechanism and protein-protein interactions. The central hypothesis is that a conformational switch in Scc4’s structure, induced by binding native ligands, regulates its dual activity. The rationale for the proposed research is that inhibitors to disrupt Scc4’s functions can be rationally designed, resulting in new approaches to treat C. trachomatis and new molecular tools to study the T3SS and developmental cycle. The central hypothesis and objectives of this application will be tested and attained by pursuing two specific aims: (1) identify small molecule ligands that bind or disrupt Scc4 and its T3SS complexes and (2) map the native protein- and small molecule-ligand binding sites on Scc4 and Scc4:Scc1 and identify allosteric effects. It is anticipated that these aims will yield the expected outcome of a structural model of ligand-binding sites on Scc4 and the Scc4:Scc1 complex from native ligands and hit-com- pounds. This outcome is expected to have an important positive impact because characterization of the protein binding sites and identification of small molecule ligands provide the information necessary to develop drugs targeting Scc4’s conformational switch or protein-protein interactions, fundamentally advancing the fields of structural biology and infectious disease biology. This contribution is significant because it is the first step in a continuum of research that is expected to lead to understanding Scc4’s dual functions at the atomic level and contribute to the treatment of the most common, sexually transmitted bacterial disease. The proposed research is innovative because the NMR structural and biochemical characterization of a unique bi-functional protein from C. trachomatis to target bacterial virulence as a therapeutic strategy represents a new and substantive departure from the status quo.