structural characterization of bacterial secretion channels

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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 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. To investigate the roles of the 4 BAM lipoproteins and how they assemble and function together, 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, as well as the use of BAM as a toxin importer in contact-dependent growth inhibition. Ongoing research 2022: 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. We recently solved the first SAM complex structures at 3.2A resolution using cryo-EM, from single particles in detergent and in lipid nanodiscs and published this work in Nature Communications in 2020. Current experiments explore the functions of the individual subunits and the folding/insertion mechanism. We recently developed a mitochondrial import assay to probe characteristics of substrates that are targeted to SAM, and we are exploring small molecule inhibitors that can bind and inactive the SAM complex. These may serve as templates for targeted therapeutics in the future. We wrote a comprehensive review on this topic in 2021. 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 its role in association with VDAC and (separately) with SAM. We wrote several reviews on these topics in 2021. This past year, we published a review on fungal outer membrane protein biogenesis in Current Opinion in Structural Biology (2022). References: 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 Bakelar, J., Buchanan, S.K. & Noinaj, N. (2016). The structure of the -barrel assembly machinery complex. Science 351:180-186. PMCID: PMC4883095 Noinaj N, Gumbart JC, Buchanan SK (2017) The -barrel assembly machinery in motion. Nat Rev Microbiol 15:197-204 PMCID: PMC5455337 Diederichs K.A., Ni X., Rollauer S.E., Botos I., Tan X., King M.S., Kunji E.R.S., Jiang J., Buchanan S.K. (2020). Structural insight into mitochondrial -barrel outer membrane protein biogenesis. Nat Commun. 2020 Jul 3;11(1):3290. doi: 10.1038/s41467-020-17144-1.PMID: 32620929 Free PMC article. Diederichs, K.A., Buchanan, S.K. & Botos, I. (2121) Building better barrels - -barrel biogenesis and insertion in bacteria and mitochondria. J Mol Biol 433(16):166894. PMCID: PMC8292188 Varughese, J. Buchanan, S.K. & Pitt, A.S. (2021). The role of Voltage-Dependent Anion Channel in mitochondrial dysfunction and human disease. Cells. 2021 Jul 9;10(7):1737. doi: 10.3390/cells10071737. PMCID: PMC8305817 Pitt, A.S. & Buchanan, S.K. (2021). A biochemical and structural understanding of TOM complex interactions and implications for human health and disease. Cells. 2021 May 11;10(5):1164. doi: 10.3390/cells10051164. PMCID: PMC8150904 Guerin, J. & Buchanan, S.K. (2021). Protein import and export across the bacterial outer membrane. Curr Opin Struct Biol, 69:55-62.doi: 10.1016/j.sbi.2021.03.007. PMCID: PMC8405454