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

我们早期的晶体结构显示了铁转运蛋白如何特异性识别与小分子（如肠杆菌素（大肠杆菌合成的铁载体）和柠檬酸盐）结合的 Fe3+。每个转运蛋白都有一个独特的结合口袋，用于其首选的小分子。当正确的底物结合时，转运蛋白会发生构象变化，通过外膜发送信号并为运输做好准备。 我们扩大了这一领域的研究，以确定脑膜炎奈瑟菌如何与人血清转铁蛋白结合并提取铁以输入细菌细胞。这些细菌需要铁才能生存，并直接从人类蛋白质中获取铁。奈瑟氏球菌具有外膜蛋白 TbpA 和辅助受体蛋白 TbpB，它们一起可以从称为转铁蛋白的人类血浆蛋白中提取铁。我们综合运用 X 射线晶体学、电子显微镜、小角 X 射线散射、生物化学和分子动力学模拟来阐明铁清除机制。这是细菌外膜蛋白与其全长人类靶蛋白结合的第一个原子分辨率结构。 在寻找新型抗菌疗法的过程中，我们扩展了对小分子转运蛋白的研究，以探究蛋白质如何穿过外膜。我们研究的一些金属转运蛋白还促进称为大肠菌素的大型蛋白质毒素的摄取。例如，我们确定了鼠疫耶尔森氏菌（引起鼠疫）的毒力所需的外膜铁转运蛋白的结构。我们还确定了一种称为害虫素的大肠菌素的结构，它利用这种转运蛋白穿过外膜。这两种结构向我们展示了如何设计一种新型抗生素，这是针对任何革兰氏阴性细菌的噬菌体疗法的第一个例子，并且我们的抗生素被证明对临床分离株有效。在这一成功的指导下，我们将继续这种类型的蛋白质工程对于其他细菌病原体。 有趣的是，对于所有这些过渡金属转运蛋白，金属如何进入周质尚不清楚。我们知道，运输涉及内膜蛋白复合物 (TonB-ExbB-ExbD) 和质子动力形式的能量。我们最近确定了该运动的子复合体的结构，由 ExbB 和 ExbD 组成。我们使用 X 射线晶体学、电子显微镜、DEER 光谱、交联和电生理学的组合方法来证明 Ton 子复合物形成 pH 敏感的阳离子选择性通道，将离子流与外膜上的能量转导耦合起来。 2020年持续工作 另一种对 NIH 临床中心非常重要的医院获得性感染是肺炎克雷伯菌。这种细菌表现出多重耐药性，一些菌株表现出高毒力。为了寻找对抗感染的新方法，我们正在与 NCI 的 Susan Gottesman 合作，研究参与荚膜调节的蛋白质。肺炎克雷伯菌可以逃避免疫检测，并以其围绕外膜的厚胶囊层阻止抗生素的渗透。我们的假设是，荚膜合成的下调可能会使肺炎克雷伯菌对可用的抗生素更加敏感，因此比目前的情况更容易治疗。该系统的结构和功能实验正在进行中。 在一个针对肺炎克雷伯菌的单独项目中，我们最近确定了 Kp 需氧菌素转运蛋白的四种结构，它是一种依赖于 TonB 的转运蛋白，与高毒力肺炎克雷伯菌的毒力相关。我们目前正在使用计算机药物筛选和 STD-NMR 来识别与 aerobactin 竞争结合的小分子，并计划在该疾病的动物模型中探索这些化合物。这项工作将于明年完成并出版。 根据 2016 年《自然》杂志发表的有关 Ton 运动子复合体的研究成果，我们最近通过冷冻电镜解析了脂质纳米盘中这种纳米机器的 3.3 A 结构，以回答与化学计量、亚基排列和功能相关的问题。虽然我们发现 ExbBD 的亚基化学计量与我们 2016 年《自然》论文中的相同，但亚基的排列有所不同。这项工作发表在《自然》通讯生物学上。我们目前正在优化整个 TonB-ExbB-ExbD 复合物的样品，以便通过冷冻电镜确定结构。一旦解决，我们将能够确定能源生产的机制。最近，细菌鞭毛马达和驱动滑行运动的马达的相关结构作为预印本发表在 BioRxiv 上。它们显示出相同的排列和化学计量，符合我们的预测。我们刚刚在《结构生物学当前观点》上提交了关于该主题的评论。 参考 Buchanan, S.K.、Smith, B.S.、Venkatramani, L.、Xia, D.、Palnitkar, M.、Chakraborty, R.、van der Helm, D. 和 Deisenhofer, J. (1999)。大肠杆菌外膜主动转运蛋白 FepA ​​的晶体结构。纳特。结构。生物。 6、56-63。 Yue, W.W.、Grizot, S. 和 Buchanan, S.K. （2003）。无铁柠檬酸盐和柠檬酸铁与 TonB 依赖性外膜转运蛋白 FecA 结合的结构证据。 J.莫尔。生物。 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. ＆埃塞尔，L.（2007）。与大肠菌素 Ia R 结构域结合的大肠菌素 I 受体的结构：对蛋白质输入的影响。 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. 和 Buchanan, S.K. （2012）。致病性奈瑟菌盗铁的结构基础。自然 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. 和 Buchanan， S.K. （2012）。针对革兰氏阴性病原体的噬菌体溶素的结构工程。过程。国家。阿卡德。科学。美国，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. 和 Buchanan, S.K. （2016）。革兰氏阴性病原体 LPS 转运蛋白 LptDE 的结构和功能特征。结构 24：965-76。 PMCID：PMC4899211 Celia, H.、Noinaj, N.、Zakharov, S.D.、Bordignon, E.、Botos, I.、Santamaria, M.、Cramer, W.A.、Lloubes, R. 和 Buchanan, S.K. （2016）。从结构角度了解 Ton 复合物在能量传导中的作用。自然 538：60-65。 PMCID：PMC5161667 Celia, H.、Botos, I.、Ni, X.、Fox, T.、De Val, N.、Lloubles, R.、Jiang, J. 和 Buchanan, S.K. （2019）。细菌 Ton 运动子复合体 ExbB-ExbD 的冷冻电镜结构提供了结构和化学计量的信息。 Commun Biol 10 月 4 日；2:358。 DOI：10.1038/s42003-019-0604-2。 eCollection 2019。PMCID：PMC6778125