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. Similar mechanisms for OMP biogenesis exist for mitochondria and chloroplasts, where interaction of the bacterial or mitochondrial signal sequence located in the final transmembrane beta strand associates with the first beta strand of BamA/Sam50, followed by strand integration into the BAM/SAM complex. We solved structures of BamA (2013), the BAM complex (2016), and the SAM complex (2021). However, even with these structures being known, the mechanism for how the BAM or SAM complex recognizes, folds, and inserts nascent OMPs into the outer membrane remains to be determined. We wrote a comprehensive review on this topic in 2021, while in 2022, we published a review on fungal outer membrane protein biogenesis in Current Opinion in Structural Biology (2022). Ongoing research 2025: With the successful structure determination of all components of the BAM complex, we focused on the mitochondrial homolog, the Sorting and Assembly Machinery, SAM complex. While Sam50 and BamA are both essential proteins and are predicted to be structural and functional homologs, the peripheral components of the SAM complex, Sam35 and Sam 37, are completely unrelated to BamB, BamC, BamD, and BamE. Structural and functional characterization of the SAM complex components sheds light on how mitochondria have evolved to insert proteins into the mitochondrial outer membrane. We recently solved the first SAM complex structures at 3.2Å resolution using cryo-EM, both from single particles in detergent and in lipid nanodiscs. We published this work in Nature Communications in 2020. We wrote a comprehensive review on this topic in 2021. Current experiments explore the functions of the individual subunits and the folding/insertion mechanism. We 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 inactivate the SAM complex. These may serve as templates for targeted therapeutics in the future. For example, we recently solved two 2.8Å cryo-EM structures of the Thermothelomyces thermophilus SAM complex in the absence of substrate in which the Sam50 β-barrel adopts two different conformations; the first is a closed barrel as observed in previously published structures, while the second contains a Sam50 with the first four β-strands rotated outwards resulting in an open barrel. To understand how these dynamics are influenced by substrate, we studied the interaction of the SAM complex with a β-signal peptide mimic, darobactin A[14]. Darobactin A binds to the SAM complex with nanomolar affinity and inhibits the import and assembly of mitochondrial β-barrel proteins in vitro. We also determined a 3.0Å cryo-EM structure of the Thermothelomyces thermophilus SAM complex bound to darobactin A, revealing that darobactin A stabilizes an open Sam50 lateral gate conformation by binding to strand β1, therefore blocking β-barrel biogenesis. This work is in revision at Nature Communications, with expected publication within the new few months. 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 Diederichs, K.A., Pitt, A.S., Varughese, J.T., Hackel, T.N. Buchanan, S.K. & Shaw, P.L. (2022). Mechanistic insights into fungal mitochondrial outer membrane protein biogenesis. Curr Opin Struct Biol, 2022 Jun;74:102383. doi: 10.1016/j.sbi.2022.102383. Epub 2022 Apr 30. PMCID: PMC9189057