Structure Determination Of Bacterial Outer Membrane Prot

细菌外膜蛋白结构测定

• 批准号: 7151490
• 负责人: SUSAN K. BUCHANAN
• 金额: --
• 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/7151490
• 关键词: Escherichia coli; Neisseria meningitidis; X ray crystallography; bacterial meningitis; bacterial proteins; catechols; cell surface receptors; colicines; crystallization; ion transport; iron; membrane transport proteins; methionine; microcalorimetry; microorganism growth; protein protein interaction; protein structure; protein structure function; receptor binding; siderophores; stoichiometry; structural biology; transferrin

Transport in Gram-negative organisms is initiated by passage of the transported species through a transmembrane beta-barrel in the outer membrane. The transport of iron is particularly important for bacterial growth, and outer membrane iron transporters are therefore major vaccine targets against pathogens such as Neisseria, Haemophilus, and Yersinia. These transporters show high affinity and specificity for Fe(III)-ligand complexes, and require energy derived from the proton motive force across the inner membrane to transport ferric complexes. The required energy is provided by transient interaction with an integral inner membrane protein complex, TonB-ExbB-ExbD, resulting in a protein assembly that spans both the inner and outer membranes, as well as the periplasmic space. During 2004, we worked on the following projects: [1] Alternate mechanisms of ligand binding: structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA: We recently solved the structures of FecA, the E. coli ferric citrate transporter, alone and in complex with dicitrate and diferric dicitrate, to resolutions of 2.1 A to 3.4 A. These three structures together shed new light on how TonB-dependent transporters recognize and bind their cognate ligands. We showed for the first time that FecA can bind both iron-free and ferric ligands, and that binding an iron-free ligand is a physiologically relevant phenomenon. The structure of FecA bound with iron-free dicitrate represents the first structure of a TonB-dependent transporter bound with an iron-free siderophore. We deduced the structural mechanism for discrimination between the iron-free and ferric siderophore: the binding of diferric dicitrate, but not iron-free dicitrate alone, causes major conformational rearrangements in the transporter. From these data we proposed a new model for ligand binding with implications for vaccine design: FecA binds iron-free dicitrate in the non-productive state or first step, followed by siderophore displacement or iron extraction to form the transport-competent, diferric dicitrate bound state in the second step. It is important to understand the complexities of ligand binding in order to develop vaccines or chemotherapeutic agents that target this family of transporters. Our discovery that FecA can bind an iron-free ligand led to a collaboration on outer membrane transporters that can bind iron-free ligands. [2] Plug domain folding and ability to bind ligand: the plug domain of a Neisserial TonB-dependent transporter retains structural integrity in the absence of its transmembrane beta-barrel: Neisseria meningitidis is the causative agent of bacterial meningitis. This blood-borne pathogen acquires iron from human transferrin (hTf = 80 kDa) through an outer membrane transporter complex, transferrin binding proteins A and B (TbpA = 100 kDa; TbpB = 68-85 kDa). TbpA and TbpB form a discrete complex to bind transferrin synergistically, yet each protein is also capable of binding transferrin on its own. TbpA is a TonB-dependent outer membrane iron transporter. We are interested in learning how this human pathogen can extract iron from transferrin and transport it into the periplasm. To evaluate the contribution of the plug domain to ligand recognition and binding, and to investigate its stability in the absence of the transmembrane beta-barrel, the N-terminal 160 residues (plug domain) of TbpA were overexpressed in both the periplasm and cytoplasm of E. coli. We found this domain to be soluble and monodisperse in solution, exhibiting secondary structure elements found in plug domains of structurally characterized TonB-dependent transporters, such as FepA and FecA. Although the TbpA plug domain is apparently correctly folded, we were not able to observe an interaction with human transferrin by isothermal titration calorimetry or nitrocellulose binding assays. This work contrasts with similar experiments performed on the plug domain of FepA, which was found to be unfolded but capable of binding ligand with a 100-fold reduced affinity. Our experiments suggest that the plug domain may fold independently of the beta-barrel, but extracellular loops of the beta-barrel are required for binding transferrin. [3] Maintenance of the cell envelope by outer membrane-associated proteins: structure of the OmpA-like domain of RmpM from Neisseria meningitidis: We recently solved the 1.9 A crystal structure of the C-terminal (periplasmic) domain of N. meningitidis RmpM, a protein that has been shown to interact with integral outer membrane proteins and that contains a domain responsible for non-covalent peptidoglycan binding. This domain is widely conserved across Gram-negative bacteria, with family members including the peptidoglycan-associated lipoprotein PAL and the flagellar motor protein MotB; ours is the first structure of such a domain. Although RmpM was predicted to be an outer membrane protein, our structure suggests that RmpM instead associates with integral outer membrane proteins through its N-terminal domain, while its C-terminal domain binds peptidoglycan. In this manner, RmpM can stabilize the cell envelope. From the structure of the OmpA-like domain of RmpM, we suggested a putative peptidoglycan binding site and identified residues that may be essential for binding. Both the crystal structure and solution experiments indicated that RmpM may exist as a dimer. This would promote more efficient peptidoglycan binding, by allowing RmpM to interact simultaneously with two glycan chains through its C-terminal, OmpA-like binding domain, while its (structurally uncharacterized) N-terminal domain could stabilize oligomers of porins and TonB-dependent transporters (such as TbpA) in the outer membrane.

革兰氏阴性微生物中的转运是由转运的物质通过外膜中的跨膜β-桶而启动的。铁的转运对于细菌生长特别重要，因此外膜铁转运蛋白是针对病原体如奈瑟氏球菌、嗜血杆菌和耶尔森氏菌的主要疫苗靶标。这些转运蛋白对Fe（III）-配体复合物表现出高亲和力和特异性，并且需要来自跨内膜质子动力的能量来转运铁复合物。所需的能量通过与完整的内膜蛋白复合物TonB-ExbB-ExbD的瞬时相互作用提供，从而产生跨越内膜和外膜以及周质空间的蛋白质组装。2004年，我们开展了以下项目： [1]配体结合的替代机制：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： 我们最近解决了FecA的结构，E。大肠杆菌柠檬酸铁转运蛋白，单独的和与二柠檬酸和二柠檬酸铁的复合物，至2.1 A至3.4 A的分辨率。这三种结构共同揭示了TonB依赖性转运蛋白如何识别和结合其同源配体。我们首次表明，FecA可以结合无铁和铁配体，并且结合无铁配体是一种生理相关现象。与无铁二柠檬酸盐结合的FecA的结构代表与无铁铁载体结合的TonB依赖性转运蛋白的第一结构。我们推断的结构机制之间的无铁和铁的铁载体的歧视：二铁二柠檬酸盐的结合，但不单独无铁二柠檬酸盐，导致主要的构象重排的转运。从这些数据中，我们提出了一个新的配体结合模型与疫苗设计的影响：FecA结合无铁的二柠檬酸在非生产状态或第一步，其次是铁载体置换或铁提取，以形成运输能力，二柠檬酸二铁结合状态的第二步。重要的是要了解配体结合的复杂性，以便开发针对该转运蛋白家族的疫苗或化疗剂。我们发现FecA可以结合无铁配体，这导致了可以结合无铁配体的外膜转运蛋白的合作。 [2]塞结构域折叠和结合配体的能力：奈瑟球菌TonB依赖性转运蛋白的塞结构域在不存在其跨膜β-桶的情况下保持结构完整性： 脑膜炎奈瑟菌是细菌性脑膜炎的病原体。这种血液传播的病原体通过外膜转运蛋白复合物、转铁蛋白结合蛋白A和B（TbpA = 100 kDa; Tbp B = 68-85 kDa）从人转铁蛋白（hTf = 80 kDa）获得铁。TbpA和TbpB形成离散复合物以协同地结合转铁蛋白，然而每种蛋白质也能够自身结合转铁蛋白。TbpA是一种依赖于TonB的外膜铁转运蛋白。我们有兴趣了解这种人类病原体如何从转铁蛋白中提取铁并将其运输到周质中。为了评估plug结构域对配体识别和结合的贡献，并研究其在缺乏跨膜β-桶的情况下的稳定性，将TbpA的N-末端160个残基（plug结构域）在E.杆菌我们发现这个域是可溶的，在溶液中是单分散的，表现出二级结构元素中发现的结构特征的TonB依赖性转运蛋白，如FepA和FecA的插头域。虽然TbpA插头域显然是正确折叠，我们无法观察到与人转铁蛋白的相互作用，通过等温滴定量热法或硝酸纤维素结合试验。这项工作与在FepA的插入结构域上进行的类似实验形成对比，发现FepA是未折叠的，但能够以100倍降低的亲和力结合配体。我们的实验表明，塞域可以折叠独立的β-桶，但细胞外环的β-桶所需的结合转铁蛋白。 [3]外膜相关蛋白对细胞包膜的维持：脑膜炎奈瑟菌RmpM的OmpA样结构域的结构： 我们最近解决了1.9 A晶体结构的C-末端（周质）域的N。脑膜炎RmpM，一种已显示与完整外膜蛋白相互作用并含有负责非共价肽聚糖结合的结构域的蛋白。该结构域在革兰氏阴性细菌中广泛保守，其家族成员包括肽聚糖相关脂蛋白PAL和鞭毛马达蛋白MotB;我们的是这种结构域的第一个结构。虽然RmpM被预测为外膜蛋白，但我们的结构表明RmpM通过其N-末端结构域与完整的外膜蛋白结合，而其C-末端结构域与肽聚糖结合。以这种方式，RmpM可以稳定细胞包膜。从RmpM的OmpA样结构域的结构，我们提出了一个假定的肽聚糖结合位点，并确定了可能是必不可少的结合残基。晶体结构和溶液实验均表明RmpM可能以二聚体形式存在。这将促进更有效的肽聚糖结合，通过允许RmpM通过其C-末端OmpA样结合结构域同时与两条聚糖链相互作用，而其（结构上未表征的）N-末端结构域可以稳定外膜中的孔蛋白和TonB依赖性转运蛋白（如TbpA）的寡聚体。