A molecular understanding of bacterial virulence factors is critical to address the urgent need for effective new vaccines and therapeutics for increasingly dangerous antibiotic resistant microbes, emerging diseases and bio-terrorism threats. Type IV pill (T4P) are key virulence factors for Gram negative bacteria with diverse roles in surface motility, adhesion, microcolony and biofilm formation, signal transduction, and DNA transformation. These surface-exposed T4P are targets for the host immune response, as well as for vaccines and therapeutic reagents. The T4P system is structurally related to the Type II secretion system (T2SS), which exports toxins in pathogenic bacterial species, and to the archaeal flagellar system, needed for motility. These three systems employ multiple protein components to build multifunctional filaments spanning the periplasmic space. To achieve detailed characterizations of such challenging filament systems, we will integrate biochemical, biophysical and structural characterizations by both x-ray crystallography and electron microscopy. These experiments aim to solve a prototypical set of subunit and filament structures, and to define assembly ATPase conformations and their complexes with membrane protein partners. We will work outward from the subunit cores to the conformational states and interaction domains and then to characterize interfaces and protein:protein complexes. This joint Tainer-Craig proposal will thus provide an integrated visualization built up from the individual protein domains to the complex T4P and T2SS systems and thereby fill a critical gap in the structural biology of prokaryotic filamentous assemblies. Assembly structures will be further tested by mutagenesis and deuterium exchange-mass spectrometry. The proposed experiments will thus build a molecular description of assemblies relevant to understanding their pathogenic variation to escape the immune response, diverse biological roles, target sites for drug discovery, and design with protective epitopes for vaccines.
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