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: 8939481
• 负责人: Susan Buchanan
• 金额: $ 157.16万
• 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/8939481
• 关键词: Acinetobacter; Antibiotics; Area; Bacteria; Bacterial Outer Membrane Proteins; Bacteriophages; Binding; Biochemistry; Cells; Citrates; Clinical; Code; Communicable Diseases; Complex; Computer Simulation; Data; Detection; Down-Regulation; Drug Targeting; Electron Microscopy; Engineering; Enterobactin; Escherichia coli; Exhibits; Family; Gram-Negative Bacteria; Growth; Human; Human Genome; Immune; Infection; Integral Membrane Protein; Iron; Klebsiella pneumonia bacterium; Lead; Length; Lipid Bilayers; Lipopolysaccharides; Lower Organism; Manuscripts; Membrane; Membrane Proteins; Membrane Transport Proteins; Metals; Multi-Drug Resistance; Nature; Neisseria; Neisseria meningitidis; Nosocomial Infections; Organism; Pathway interactions; Penetration; Pesticin; Pharmacologic Substance; Plague; Plasma Proteins; Preclinical Drug Evaluation; Preparation; Protein Binding; Protein Engineering; Proteins; Reagent; Regulation; Resolution; Roentgen Rays; Serum; Siderophores; Signal Transduction; Solutions; Structure; Surface; System; Thick; Toxin; Transferrin; Transition Elements; Transport Process; United States National Institutes of Health; Vaccines; Virulence; Work; X-Ray Crystallography; Yersinia pestis; Zinc; antimicrobial; capsule; colicin; combat; interest; molecular dynamics; novel; pathogen; periplasm; preference; prevent; protein complex; protein structure; receptor; research study; small molecule; success; uptake; zinc-binding protein

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 recently 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 (Noinaj et al and Buchanan, Nature 2012). We have expanded our focus on transition metal transport to Acinetobacter baumanni zinc uptake. A. baumannii is a hospital-acquired infection demonstrating multidrug resistance. It is of great interest to the NIH clinical center. Zinc correlates with virulence in A. baumannii and there are three putative TonB-dependent zinc transporters in this bacterium. When deprived of zinc, A. baumannii becomes much more sensitive to existing antibiotics, so inhibition of zinc uptake may lead to novel therapies against this Gram-negative bacterium. We just solved the structure of an A. baumannii zinc transporter with zinc bound. We are using the structure to perform in silico small molecule drug screening and to investigate the zinc transport pathway. A manuscript describing this work is in preparation. 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 are currently working to provide structural data on the transport process. 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 iron 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 a number of clinical isolates (Lukacik et al and Buchanan, PNAS 2012). Guided by this success, we will continue this type of protein engineering for other bacterial pathogens. 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. Finally, in addition to studying outer membrane transporters, we are interested in any outer membrane protein complexes that are essential for bacterial growth, since these proteins may make good vaccine and drug targets. One such complex is LptDE, which puts newly synthesized lipopolysaccharide (LPS) into the outer membrane. In most Gram-negative bacteria, this complex is essential for viability. We recently crystallized LptDE from three different bacterial pathogens and structure solution for all three is underway. We will use these structures to develop new antimicrobial reagents.

我们早期的晶体结构显示了铁转运蛋白如何特异性地识别与小分子结合的Fe 3+，如肠杆菌素（由大肠杆菌合成的铁载体）和柠檬酸盐。 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，转运蛋白发生构象变化，通过外膜发送信号，并为转运系统做好准备。 我们最近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。这些细菌需要铁来生存，并直接从人类蛋白质中获得。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血浆蛋白中提取铁。我们使用了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相结合的方法来阐明铁清除机制。这是细菌外膜蛋白与其全长人靶蛋白结合的第一个原子分辨率结构（Noinaj等人和Buchanan，Nature 2012）。 我们已经扩大了我们的重点过渡金属转运鲍曼不动杆菌锌的吸收。A.鲍曼不动杆菌是一种医院获得性感染，表现出多重耐药性。NIH临床中心对此非常感兴趣。锌与A.在该细菌中存在三种推定的TonB依赖性锌转运蛋白。缺锌时，A.鲍曼不动杆菌对现有抗生素变得更加敏感，因此抑制锌的吸收可能会带来针对这种革兰氏阴性细菌的新型疗法。我们刚刚解出了A的结构结合锌的鲍曼不动杆菌锌转运蛋白。我们正在使用该结构进行计算机小分子药物筛选，并研究锌转运途径。描述这项工作的手稿正在编写中。 有趣的是，对于所有这些过渡金属转运蛋白，金属是如何进入周质的还没有很好的理解。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。我们目前正在努力提供运输过程的结构数据。 在寻找新型抗菌疗法的过程中，我们将小分子转运蛋白的工作扩展到了蛋白质是如何穿过外膜的。 我们研究的一些铁转运蛋白也促进了被称为大肠杆菌素的大蛋白毒素的摄取。例如，我们确定了鼠疫耶尔森氏菌（导致鼠疫）的外膜铁转运蛋白的结构，这是毒力所必需的。我们还确定了一种叫做pesticin的大肠杆菌素的结构，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工程化一种新的抗生素，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许多临床分离株有效（Lukacik等人和Buchanan，PNAS 2012）。在这一成功的指导下，我们将继续对其他细菌病原体进行这种类型的蛋白质工程。 另一个对NIH临床中心非常重要的医院获得性感染是肺炎克雷伯氏菌。这种细菌表现出多重耐药性，有些菌株表现出超强毒力。为了确定对抗感染的新方法，我们正在与NCI的Susan Gottesman合作，研究参与胶囊调节的蛋白质。K.肺炎克雷伯氏菌可以逃脱免疫检测，并通过其包围外膜的厚囊层防止抗生素渗透。我们的假设是，胶囊合成的下调可能使K。肺炎对可用的抗生素更敏感，因此比目前的情况更可治疗。该系统的结构和功能实验正在进行中。 最后，除了研究外膜转运蛋白外，我们还对细菌生长所必需的任何外膜蛋白复合物感兴趣，因为这些蛋白质可能成为良好的疫苗和药物靶点。一种这样的复合物是LptDE，它将新合成的脂多糖（LPS）放入外膜。在大多数革兰氏阴性细菌中，这种复合物对生存力至关重要。我们最近从三种不同的细菌病原体中结晶出LptDE，并且正在进行所有三种细菌病原体的结构解决方案。我们将利用这些结构来开发新的抗微生物试剂。