Structural characterization of OM proteins from Gram-negative pathogens

革兰氏阴性病原体 OM 蛋白的结构表征

• 批准号: 10248117
• 负责人: Susan Buchanan
• 金额: $ 136.16万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10248117
• 关键词: Animal Disease Models; Antibiotics; Area; Bacteria; Bacterial Outer Membrane Proteins; Bacteriophages; Binding; Binding Proteins; Biochemistry; Biology; Cations; Cells; Citrates; Clinical; Code; Colicin Ia; Communicable Diseases; Communication; Complex; Cryoelectron Microscopy; Crystallization; Detection; Down-Regulation; Drug Screening; Drug Targeting; Electron Microscopy; Electrophysiology (science); Engineering; Enterobactin; Escherichia coli; Exhibits; Family; Foxes; Gram-Negative Bacteria; Human; Human Genome; Immune; Infection; Integral Membrane Protein; Ions; Iron; Klebsiella pneumoniae; Length; Lipids; Lower Organism; Membrane; Membrane Proteins; Membrane Transport Proteins; Metals; Molecular Conformation; Motor; Movement; Multi-Drug Resistance; Nanostructures; Nature; Neisseria; Neisseria meningitidis; Nosocomial Infections; Paper; Pathogenicity; Penetration; Pesticin; Pharmacologic Substance; Plague; Plasma Proteins; Positioning Attribute; Production; Protein Engineering; Protein Import; Proteins; Publications; Publishing; Regulation; Resolution; Roentgen Rays; Role; Sampling; Serum; Siderophores; Signal Transduction; Spectrum Analysis; Structure; Surface; System; Thick; Toxin; Transferrin; Transition Elements; United States National Institutes of Health; Vaccines; Virulence; Work; X-Ray Crystallography; Yersinia pestis; aerobactin; antimicrobial; capsule; clinical center; colicin; combat; crosslink; experimental study; in silico; insight; interest; lysin; molecular dynamics; nanodisk; nanomachine; novel; pathogen; pathogenic bacteria; periplasm; prevent; protein complex; protein structure; receptor; receptor binding; small molecule; stoichiometry; structural biology; success; uptake

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. We recently 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. Ongoing work 2020 Another hospital-acquired infection of great importance to the NIH clinical center is Klebsiella pneumoniae. This bacterium exhibits multidrug resistance and some strains have shown hypervirulence. In an effort to identify new ways to combat infection, we are collaborating with Susan Gottesman, NCI, to investigate proteins involved in regulation of capsule. K. pneumoniae can escape immune detection and prevent penetration of antibiotics with its thick capsule layer that surrounds the outer membrane. Our hypothesis is that down-regulation of capsule synthesis might make K. pneumoniae more sensitive to available antibiotics, and thus more treatable than is currently the case. Structural and functional experiments on this system are in progress. In a separate project targeting Klebsiella pneumoniae, we recently determined four structures of the Kp aerobactin transporter, which is a TonB dependent transporter that correlates with virulence in hypervirulent K. pneumoniae. We are currently using in silico drug screening and STD-NMR to identify small molecules that compete for binding with aerobactin, with plans to explore these compounds in an animal model of the disease. This work will be finalized and published within the coming year. Working from our 2016 Nature publication on the Ton motor subcomplex, we recently solved the 3.3 A structure of this nano-machine in lipid nanodiscs by cryo-EM to answer questions related to stoichiometry, subunit arrangement, and function. While we find the same subunit stoichiometry for ExbBD as in our 2016 Nature paper, the arrangement of subunits differs. This work was published in (Nature) Communications Biology. We are currently optimizing samples of the entire TonB-ExbB-ExbD complex for structure determination by cryo-EM. Once solved, we will be in a position to determine the mechanism of energy production. Recently related structures from the bacterial flagellar motor and a motor that drives gliding movement were published as preprints on BioRxiv. They show the same arrangement and stoichometry, following our predictions. WE have just submitted a review at Current Opinion in Structural Biology on this topic. 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

我们的早期晶体结构显示了铁转运体如何特异性识别结合在小分子上的Fe3+，如肠杆菌蛋白（由大肠杆菌合成的铁载体）和柠檬酸盐。每个转运体都有一个独特的结合袋，用于其首选的小分子。当正确的底物结合时，转运体发生构象变化，向外膜发送信号，为运输系统做好准备。