The role of BamA in the biogenesis of beta-barrel membrane proteins

BamA 在 β-桶膜蛋白生物发生中的作用

基本信息

批准号：
9110832
负责人：
Nicholas Noinaj
金额：
$ 10.8万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-15 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9110832
关键词：
Academia Agonist Bacteria Binding Biochemical Biogenesis Cause of Death Cell Survival Cessation of life Communicable Diseases Complex Country Cytoplasm Cytoprotection Detergents Development Electron Microscopy Faculty Funding Future G-Protein-Coupled Receptors Goals Gram-Negative Bacteria Health Hemophilus ducreyi Human Immune response Infectious Agent Institution Knowledge Label Learning Length Lipoproteins Manuscripts Mediating Membrane Membrane Proteins Methods Molecular Molecular Chaperones Multi-Drug Resistance Names Nature Neisseria Neisseria gonorrhoeae Neurotensin Neurotensin Receptors Neurotransmitters Nutrient Peptidoglycan Phase Proteins Publishing Reporting Research Role Route Signal Transduction Site Spectrum Analysis Structure Surface Technology Transferrin Virulence Work X-Ray Crystallography base beta barrel career crosslink fascinate insight interest member membrane biogenesis pandemic disease pathogenic bacteria peptidomimetics periplasm protein folding receptor resistant strain success tenure track

项目摘要

DESCRIPTION (provided by applicant): Infectious diseases cause widespread sickness throughout the world each year and are the second leading cause of death, particularly in underdeveloped countries. And with the emergence of multi-drug resistance strains, the necessity for new, more effective, and more sustainable therapies is immediate and vital to protect against any future pandemics. My studies will provide crucial insight into the biogenesis of surface receptors and proteins that assist pathogenic bacteria in their virulence. This knowledge will significantly assist in the development of better therapies against these infectious agents. Gram-negative bacteria 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, signaling, and bacterial virulence. 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 BAM complex which folds and inserts the new OMPs into the outer membrane. Exactly how the BAM complex is able to accomplish its function remains unknown. However, we do know that the BAM complex consists of five subunits named BamA (an OMP itself), BamB, BamC, BamD, and BamE, which are all lipoproteins. Studies have shown that BamA and BamD are absolutely essential for cell viability and OMP biogenesis. Our lab and others have reported the structures of BamB, BamC, BamD, BamE and a large portion of the periplasmic domain of BamA, providing initial insight into how the BAM complex may function. However, even with these structures being known, the mechanism for how the BAM complex recognizes, folds, and inserts nascent OMPs into the outer membrane remains elusive, largely due to the lack of a full length BamA structure and complexes with BamA. Recently, I determined the crystal structures of a truncated BamA construct from Haemophilus ducreyi to 2.9 � and of a full length BamA construct from Neisseria gonorrhea to 3.2 �. In my proposed studies, I aim to build on this recent success to use X-ray crystallography to determine structures of BamA in complex with BamB-E, to use DEER spectroscopy and crosslinking to characterize the conformational dynamics of BamA, and to use crosslinking to explore the interactions between nascent OMPs and BamA and the other BAM components, with my goal being understand the functional role of BamA within the BAM complex. I have a strong background in X-ray crystallography and during my postdoctoral studies, have added a strong background in working with and crystallizing membrane proteins using the latest technologies such as new stabilizing detergents, bicelles, and lipidic cubic phase (LCP) methods. Recently, I solved the structures of two important surface proteins from pathogenic Neisseria and this work was published in Nature as a full research article (March 2012). For this manuscript, I also solved the structure of diferric human transferrin which research groups have been trying to solve for decades without success. More recently, I also solved the crystal structure of the agonist bound neurotensin receptor NTSR1, a GPCR responsible for binding neurotensin and other neurotransmitters. This work was published in Nature as well in October 2012 (Research Article). My current efforts are focused on studying BamA of the BAM complex and I will take this project with me as a tenure-track faculty member in academia. And with this, my most recent results, the crystal structures of BamA (H. ducreyi and N. gonorrhea), were also recently published in Nature as a full research article as well (Sept 2013). My long-term goal is to have my own research lab as a faculty member at a Research I academic institution where I can continue my research interests. This has been my lifelong ambition and while funding for academic research is more competitive now than ever, I remain dedicated to a career in research with aspirations that I will be able to establish a research group that significantly advances our current understanding of beta-barrel membrane proteins in both bacteria and humans.

描述（由申请人提供）：传染病每年在世界各地引起广泛的疾病，是第二大死亡原因，特别是在不发达国家，并且随着多重耐药菌株的出现，需要新的、更有效的和更有效的药物。更可持续的疗法对于预防未来的任何流行病至关重要，我的研究将为帮助致病菌发挥毒力的表面受体和蛋白质的生物发生提供重要的见解。革兰氏阴性细菌含有内膜和外膜，其中含有许多通常称为外膜蛋白（OMP）的β-桶蛋白，它们在革兰氏阴性细菌中发挥着重要的功能。在阴性细菌中，已知 OMP 在细胞质中合成，然后通过 Sec 易位子穿过内膜转运到周质中，一旦进入周质，伴侣就会引导新生的 OMP 穿过周质。在这里，新生的 OMP 被称为 BAM 复合物的复合物识别，该复合物折叠并将新的 OMP 插入外膜中。 BAM 复合物究竟如何实现其功能仍然存在。然而，我们确实知道 BAM 复合体由五个亚基组成，分别为 BamA（OMP 本身）、BamB、BamC、BamD 和 BamE。研究表明，BamA 和 BamD 对于细胞活力和 OMP 生物发生绝对重要，我们的实验室和其他实验室已经报道了 BamB、BamC、BamD、BamE 和 BamA 的大部分周质结构域的结构，为我们提供了初步了解。然而，即使这些结构已知，BAM 复合物如何识别、折叠并将新生 OMP 插入外膜的机制仍然存在。难以捉摸，主要是由于缺乏全长 BamA 结构和与 BamA 的复合物。最近，我确定了杜克雷嗜血杆菌 (Haemophilus ducreyi) 的截短 BamA 结构至 2.9 英寸以及淋病奈瑟氏菌 (Neisseria gonorriea) 的全长 BamA 结构至 3.2 英寸的晶体结构。在我提出的研究中，我的目标是在最近成功的基础上利用 X 射线晶体学来确定 BamA 的结构BamB-E，使用 DEER 光谱和交联来表征 BamA 的构象动力学，并使用交联来探索新生 OMP 和 BamA 以及其他 BAM 组件之间的相互作用，我的目标是了解 BamA 在 BAM 中的功能作用我在 X 射线晶体学方面拥有深厚的背景，并且在博士后研究期间，我在使用最新技术（例如新的稳定技术）处理和结晶膜蛋白方面拥有深厚的背景。最近，我解析了致病性奈瑟菌的两种重要表面蛋白的结构，这项工作作为完整的研究文章发表在《自然》杂志上（2012 年 3 月）。解决了研究小组几十年来一直试图解决的二铁人转铁蛋白的结构，但没有成功。最近，我还解决了激动剂结合神经降压素受体 NTSR1 的晶体结构，NTSR1 是一种负责结合的 GPCR。这项工作也于 2012 年 10 月发表在《自然》杂志上（研究文章）。随之而来的是，我的最新成果 BamA（杜克雷氏菌和淋病奈瑟菌）的晶体结构最近也作为完整的研究文章发表在《自然》杂志上（2017 年 9 月）。 2013）。我的长期目标是拥有自己的研究实验室，作为研究 I 学术机构的一名教员，在那里我可以继续我的研究兴趣，而现在学术研究的资助比现在更具竞争力。永远，我仍然致力于研究事业，希望能够建立一个研究小组，显着推进我们目前对细菌和人类β-桶膜蛋白的理解。