Structural characterization of OM proteins from Gram-negative pathogens

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Our early crystal structures showed how iron transporters specifically recognize Fe3+ bound to small molecules such as enterobactin (a siderophore synthesized by Escherichia coli) and citrate. Each transporter has a unique binding pocket for its preferred small molecule. When the correct substrate binds, the transporter undergoes conformational changes that send a signal across the outer membrane and prepare the system for transport. We expanded our studies in this area to determine how Neisseria meningitidis binds to human serum transferrin and extracts the iron for import into the bacterial cell. These bacteria require iron for survival and obtain it directly from human proteins. Neisseria have an outer membrane protein, TbpA, and a co-receptor protein, TbpB, which together can extract the iron from a human plasma protein called transferrin. We used a combined approach of X-ray crystallography, electron microscopy, small angle X-ray scattering, biochemistry, and molecular dynamics simulations to elucidate the iron-scavenging mechanism. This was the first atomic resolution structure of a bacterial outer membrane protein bound to its full-length human target protein. In our search for novel antimicrobial therapies, we extended our work on small-molecule transporters to ask how proteins are ferried across the outer membrane. Some of the metal transporters that we study also facilitate the uptake of large protein toxins called colicins. For example, we determined the structure of an outer membrane iron transporter from Yersinia pestis (which causes plague) that is required for virulence. We also determined the structure of a colicin, called pesticin, which uses this transporter to cross the outer membrane. The two structures showed us how to engineer a novel antibiotic that is the first example of phage therapy for any Gram-negative bacterium, and our antibiotic was demonstrated to be effective on clinical isolates Guided by this success, we will continue this type of protein engineering for other bacterial pathogens. Interestingly, for all of these transition metal transporters, how the metal gets into the periplasm is not well understood. We know that transport involves an inner membrane protein complex (TonB-ExbB-ExbD) and energy in the form of protonmotive force. In 2016, we determined the structure of a subcomplex of this motor, consisting of ExbB and ExbD. We used a combined approach of X-ray crystallography, electron microscopy, DEER spectroscopy, crosslinking, and electrophysiology to show that the Ton subcomplex forms pH sensitive, cation selective channels that couple ion flow to energy transduction at the outer membrane. In 2019, we used cryo-electron microscopy to determine higher resolution structures of the sub-complex with a correct stoichiometry of 5 ExbB to 2 ExbD. Furthermore, our structure of this nano-machine in lipid nanodiscs answers questions related to stoichiometry, subunit arrangement, and function. We recently solved structures of four additional species of ExbBD to determine conserved and contrasting features, which will help identify elements and sequences important for function. For example, currently the pathway for proton translocation is unclear, but the new structures combined with computational analysis will provide the foundational basis to answer this question (Ratliff, Kwon, and Buchanan, manuscript in preparation). To understand how the Ton motor complex, and related motor complexes, translate the proton motive force across the inner membrane to outer membrane complexes residing about 150Å away, we have just determined the tripartite structures of TonB-ExbBD and TolAQR. Our high-resolution structures determined by cryo-EM reveal the inner membrane-embedded engine parts of the Ton and Tol systems, showing how TonB and TolA interact with ExbBD and TolQR, respectively. Structural and sequence similarities between the two motor complexes suggest a common mechanism for opening of the proton channel and propagation of proton motive force into movement for the TonB or TolA subunits. We found that TonB and TolA bind at preferential ExbB or TolQ subunits and propose a new mechanism of motor assembly, as well as insight into their mechanism of action. So, 50+ years after TonB was discovered, we are now in a position to determine how these motors work in much greater detail. In collaboration with Susan Gottesman (NCI), we are completing structural characterization of the Regulation of Capsule Synthesis pathway in Klebsiella pneumoniae. These structures and accompanying genetic data offer new avenues of antibiotic development, which we are exploring through screening small libraries and in silico drug discovery (Nune and Buchanan, unpublished). In collaboration with John Dekker (NIAID), we are characterizing a three-component bacterial efflux pump implicated in virulence in Pseudomonas aeruginosa. Dr. Dekker has sequenced patient isolates from Pseudomonas infections indicating mutations in the pump, and our structures will further inform antibiotic resistance and drug development (Putti, Ratliff, and Buchanan, unpublished). We continue to solve structures of TonB-dependent transporters from Acinetobacter baumanii and Klebsiella pneumoniae correlated with virulence, transporting either zinc or iron (Mauraiks and Buchanan, unpublished). With our new structures of TonB-ExbBD and TolAQR motors, we are designing functional and computational experiments to determine the molecular mechanism of force generation. A deeper understanding of these bacterial motors will lead to new drug targets (Celia and Buchanan, unpublished). References Buchanan, S.K., Smith, B.S., Venkatramani, L., Xia, D., Palnitkar, M., Chakraborty, R., van der Helm, D. & Deisenhofer, J. (1999). Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat. Struct. Biol. 6, 56-63. Yue, W.W., Grizot, S. & Buchanan, S.K. (2003). Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. J. Mol. Biol. 332, 353-368. Buchanan, S.K., Lukacik, P., Grizot, S., Ghirlando, R., Ali, M.M.U., Barnard, T.J., Jakes, K.S., Kienker, P.K. & Esser, L. (2007). Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import. EMBO J. 26, 2594-2604. PMCID: PMC1868905 Noinaj, N., Easley, N.C., Oke, M., Mizuno, N., Gumbart, J., Boura, E., Steere, A., Zak, O., Aisen, P., Tajkhorshid, E.M., Evans, R., Gorringe, A., Mason, A.B., Steven, A. & Buchanan, S.K. (2012). Structural basis for iron piracy by pathogenic Neisseria. Nature 483, 53-58. PMCID: PMC3292680 Lukacik, P., Barnard, T.J., Keller, P.W., Chaturvedi, K., Seddiki, N., Fairman, J.W., Noinaj, N., Kirby, T.L., Henderson, J.P., Steven, A.C., Hinnebusch, B.J. & Buchanan, S.K. (2012). Structural engineering of a phage lysin that targets Gram-negative pathogens. Proc. Natl. Acad. Sci. USA, 109, 9857-9862. PMCID: PMC3382549 Mayclin, S.J., McCarthy, J.G., Botos, I., Lundquist, K., Majdalani, N., Wojtowicz, D., Barnard, T.J., Gumbart, J.C. & Buchanan, S.K. (2016). Structural and functional characterization of the LPS transporter LptDE from Gram-negative pathogens. Structure 24:965-76. PMCID: PMC4899211 Celia, H., Noinaj, N., Zakharov, S.D., Bordignon, E., Botos, I., Santamaria, M., Cramer, W.A., Lloubes, R. & Buchanan, S.K. (2016). Structural insight into the role of the Ton complex in energy transduction. Nature 538:60-65. PMCID: PMC5161667 Celia, H., Botos, I., Ni, X., Fox, T., De Val, N., Lloubles, R., Jiang, J., & Buchanan, S.K. (2019). Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry. Commun Biol Oct 4;2:358. doi: 10.1038/s42003-019-0604-2. eCollection 2019. PMCID: PMC6778125 Celia