Gram-negative bacteria, mitochondria, and chloroplasts contain an inner and outer membrane. The outer membrane contains a host of beta-barrel proteins commonly called outer membrane proteins (OMPs), which serve essential functions in cargo transport and signaling and are also vital for membrane biogenesis. In Gram-negative bacteria, it is known that OMPs are synthesized in the cytoplasm and then transported across the inner membrane into the periplasm via a Sec translocon. Once in the periplasm, chaperones guide the nascent OMPs across the periplasm and peptidoglycan to the inner surface of the outer membrane. Here, the nascent OMPs are recognized by a complex known as the beta-barrel assembly machinery (BAM) complex which folds and inserts the new OMPs into the outer membrane. Exactly how the BAM complex is able to accomplish its function remains unknown. However, we do know that the BAM complex consists of five components named BamA (an OMP itself) and BamB, BamC, BamD, and BamE, which are all accessory lipoproteins. Studies have shown that BamA and BamD are absolutely essential for cell viability and OMP biogenesis. Similar mechanisms for OMP biogenesis exist for mitochondria and chloroplasts, providing further evidence of the evolutionary relationship of these organelles to bacteria. In 2012, we solved the structure of BamB, while other groups solved BamC, BamD, BamE and a large portion of the periplasmic domain of BamA. Together these structures provided insight into how the BAM complex may recognize nascent OMPs. However, even with these structures being known, the mechanism for how the BAM complex recognizes, folds, and inserts nascent OMPs into the outer membrane remained elusive. To understand the mechanism of the BAM complex, we have determined crystal structures of the core membrane component called BamA, a beta-barrel membrane protein itself, from two different species (Neisseria gonorrhoeae and Haemophilus ducreyi). The structure of BamA contains a large N-terminal periplasmic domain and a C-terminal 16-stranded beta-barrel domain. The periplasmic domain was found in two different conformations representing open and closed states, which may serve as a gating mechanism to allow substrate access to the internal barrel cavity. Interestingly, the closed state was accompanied by a significant destabilization of the terminal beta strand, which was found tucked inside the barrel domain. MD simulations revealed that BamA could destabilize the local membrane along the terminal strand, thinning the membrane by as much as 16 Angstroms. In addition, these MD simulations also revealed that the barrel domain of BamA can undergo a lateral opening to create a portal from the periplasm directly into the outer membrane. This work was published in Nature in 2013, with follow-up experiments confirming that lateral opening of the beta barrel is required for BAM function published in Structure, 2014. Current experiments investigate the roles of the 4 BAM lipoproteins and how they assemble and function together. Toward this end, we published the structure of the BAM complex in Science in 2016. We are also investigating the potential of BamA to serve as a drug target for the development of novel antibiotics, since it is an essential protein in all Gram-negative bacteria. Ongoing research 2018: A recent extension of the BAM work is a new project to understand bacterial contact dependent growth inhibition, in which a bacterial two-partner secretion system binds to a BamA on a target bacterial cell for transfer of the toxin and eventual cell death. We have just solved the structure of the outer membrane transporter for the secretion partner and are currently working to structurally and functionally characterize the entire system. If BamA plays an active role in toxin import, this may illustrate new ways to target antibiotics. With the successful structure determination of all components of the BAM complex, we are now focusing on the mitochondrial homolog, the Sorting and Assembly Machinery, SAM complex. While Sam50 and BamA are predicted to be structural and functional homologs, the peripheral components of the SAM complex are completely unrelated to BamB, C, D, and E. Structural and functional characterization of the SAM complex components will shed light on how mitochondria have evolved to insert proteins into the mitochondrial outer membrane. During the past year, we have made significant progress in expression and purification of Sam50 homologs; crystallization experiments are in progress. Data collection using cryo-EM is also in progress. Another protein complex that handles mitochondrial proteins (including the outer membrane proteins destined for the SAM complex), is the Translocase of the Outer Membrane, TOM complex. We are working on structural and functional characterization of this complex, as well as the mitochondrial outer membrane protein Mdm10, which is involved in Tom40 biogenesis and lipid transfer from the endoplasmic reticulum. References: Noinaj, N., Fairman, J.W. & Buchanan, S.K. (2011). The crystal structure of BamB suggests interactions with BamA and its role within the BAM complex. J. Mol. Biol., 407, 248-260. PMCID: PMC3048904 Noniaj, N., Kuszak, A.J., Gumbart, J.C., Lukacik, P., Chang, H., Easley, N.C., Lithgow, T. & Buchanan, S.K. (2013). Structural insight into the biogenesis of beta barrel membrane proteins. Nature 501: 385-390. PMCID:PMC3779476 Noinaj, N., Kuszak, A.J., Balusek, C. Gumbart, J.C. & Buchanan, S.K. (2014). Lateral opening and exit pore formation are required for BamA function. Structure 22:1055-62. PMCID: PMC4100585 Kuszak, A.J., Jacobs, D., Gurnev, P.A., Shiota, T., Louis, J., Lithgow, T., Bezrukov, S.M., Rostovtseva, T.K & Buchanan, S.K. (2015). Evidence of distinct channel conformations and substrate binding affinities for the mitochondrial outer membrane protein translocase pore Tom40. J. Biol. Chem. 290:26204-17. PMCID: PMC4646270 Bakelar, J., Buchanan, S.K. & Noinaj, N. (2016). The structure of the -barrel assembly machinery complex. Science 351:180-186. PMCID: PMC4883095