Project Summary Bacterial pathogens membrane proteins are important, and sometimes critical, to pathogenesis and, thus, human health. Despite the importance of these proteins in regulating host interactions, they are less understood than channels, transporters and GPCRs. The study of these proteins can, therefore, provide new knowledge about membrane protein structure, function, and dynamics, as well as have impact on combatting bacterial infections. These studies are particularly important because bacterial membrane proteins are frequent targets of antibiotics for which resistance is a growing threat. This MIRA application outlines our recent endeavors in understanding two bacterial membrane proteins: Opa and LspA, each of which is independently important for bacterial proliferation in humans. The overall goal of this proposal is to determine how these proteins look (beyond static structure), how they work, and how we can use this structure/function knowledge to advance our understanding of bacterial pathogenesis and human health. The outer membrane Opa proteins from Neisseria gonorrhoeae and N. meningitidis bind various host receptors and induce bacterial engulfment into human host cells. We have previously determined the monomeric structure of Opa60 and established that Opa-reconstituted liposomes engage in receptor-mediated phagocytosis. Future directions focus on a multidisciplinary approach to understanding the oligomeric structure of Opa60 and the Opa60-receptor complex. In another avenue of research, we focus on LspA, a membrane-embedded signal peptidase that processes essential lipoproteins in nearly all bacteria. Despite the relevance of LspA as a target for antibiotics, inhibitors of LspA are currently unavailable in the clinic. Interactions between LspA and its substrate differ among bacterial species, and we aim to investigate the structure and conformational changes of LspA with substrate and investigate the molecular determinants for substrate selectivity. Finally, this proposal outlines an emerging project that investigates bacterial membrane protein – lipid interactions. Bacterial inner membrane lipid composition varies significantly between bacterial species and the hypothesis that membrane proteins evolve and adapt to these lipid composition is tested.Results of the proposed aims will provide the molecular determinants of (i) neisserial Opa – Opa and Opa – host receptor interactions, (ii) substrate binding and recognition of LspA and (iii) membrane protein – lipid interactions. This knowledge will enhance our understanding of host-pathogen interactions and aid in combatting infections associated with bacterial pathogens.