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Structure Determination Of Bacterial Outer Membrane Prot

细菌外膜蛋白结构测定

基本信息

批准号：
7337562
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
项目类别：
财政年份：
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/7337562
关键词：
Structure Determination Bacterial Outer Membrane

项目摘要

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 FY06, we published work on the following project: 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. We have separately expressed and purified TbpA and TbpB in preparation for studying the triple TbpA-TbpB-hTf complex. This work is summarized in annual reports from earlier years. In order to progress with our goal of determining the crystal structure of the triple complex, we first needed to solve the structure of human serum transferrin, a protein which has eluded structure determination for over 30 years. Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependant process. The binding and release of iron results in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. The structure of human serum transferrin (hTF) lacking iron (apo-hTF) which was independently determined by two methods: (1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7 ? resolution using a MAD phasing strategy, by substituting the nine methionines in hTF with selenomethionine and (2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 ? by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human TF and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 62? and 52? rotations are required to open the N- and C-lobe, respectively. Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. Once the full-length hTF structure was available (reference 3 in publication list), we were able to do a structural comparison of hTF and human lactoferrin (reference 4 in publication list). Questions arise as to how TbpA can distinguish hTF from lactoferrin, since the protein share high sequence similarity and bind iron in similar ways. TpbA, however, only binds (and uses iron from) hTF; it does not even bind other closely related TFs. Some of the factors influencing the recognition are identified as follows: Comparison of the structures of iron-free hTF and LTF has revealed several distinctions that could be important in the differing iron and receptor binding properties of these two proteins. Though hTF and LTF are overall quite similar in sequence and structure, they differ in the structure of their inter-lobe linker (helical in LTF and unstructured in hTF), the presence of a salt bridge between the helical linker of LTF and its C-lobe which is absent in hTF, their pattern of disulfide bonding (inter-subdomain bonding in hTF but not in LTF), the relative orientation of their lobes to one another (the C-lobe of LTF is rotated closer to the N-lobe as compared to hTF), the dilysine trigger and triad residues in hTF which are not present in LTF, the openness of their C-lobes (being more open in hTF), the structure of the C-lobe hinge regions (unstructured in hTF and β-strands in LTF), and their inter-lobe interactions (salt bridge between the C-terminal helix and N-lobe of hTF which is not found in LTF). Analysis of these differences increases our understanding of the divergent functions of these two proteins, as well the necessity for pathogenic bacteria to express independent receptors for two such similar proteins. Crystal structures of the bacterial receptors in complex with their host substrates should provide further insight into these interactions.

革兰氏阴性生物体中的转运是由转运物种通过外膜中的跨膜 β 桶启动的。铁的转运对于细菌生长尤其重要，因此外膜铁转运蛋白是针对奈瑟氏菌、嗜血杆菌和耶尔森氏菌等病原体的主要疫苗靶标。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，并且需要来自穿过内膜的质子动力的能量来转运三价铁复合物。所需的能量是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的，从而形成跨越内膜和外膜以及周质空间的蛋白质组装体。 2006 财年期间，我们发布了以下项目的工作：脑膜炎奈瑟菌是细菌性脑膜炎的病原体。这种血源性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；TbpB = 68-85 kDa) 从人转铁蛋白 (hTf = 80 kDa) 获取铁。 TbpA 和 TbpB 形成一个离散的复合物，协同结合转铁蛋白，但每种蛋白质也能够单独结合转铁蛋白。 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转运到周质中。我们分别表达并纯化了TbpA和TbpB，为研究TbpA-TbpB-hTf三重复合物做准备。这项工作在前几年的年度报告中进行了总结。为了实现确定三重复合物晶体结构的目标，我们首先需要解析人血清转铁蛋白的结构，这是一种 30 多年来一直未能确定结构的蛋白质。血清转铁蛋白可逆地结合两个叶中的铁，并通过受体介导的 pH 依赖性过程将其递送至细胞。铁的结合和释放导致巨大的构象变化，其中每个叶中的两个子域以围绕铰链的刚性扭转运动关闭或打开。通过两种方法独立测定人血清转铁蛋白（hTF）缺铁（apo-hTF）的结构：（1）在2.7℃下解析重组非糖基化apo-hTF的晶体结构。使用 MAD 定相策略，通过用硒代蛋氨酸取代 hTF 中的九个蛋氨酸，并且 (2) 确定糖基化 apo-hTF（从血清中分离）的结构，分辨率为 2.7 ?通过使用人 apo-N-lobe 和兔 Holo-C1-子域作为搜索模型进行分子替换。这两种晶体结构本质上是相同的。它们代表了第一个发表的全长人类 TF 模型，并揭示了与家族成员（人类乳铁蛋白和母鸡卵转铁蛋白）相比，两个叶几乎同样开放：62？和52？分别需要旋转才能打开 N 瓣和 C 瓣。这种结构的可用性对于全面了解 hTF 每个叶的金属结合特性至关重要； apo-hTF 结构表明 N 叶和 C 叶铰链区的差异可能会影响铁结合和释放的速率。一旦获得全长 hTF 结构（出版物列表中的参考文献 3），我们就能够对 hTF 和人乳铁蛋白进行结构比较（出版物列表中的参考文献 4）。问题在于 TbpA 如何区分 hTF 和乳铁蛋白，因为这两种蛋白质具有高度的序列相似性并以相似的方式结合铁。然而，TpbA 只结合 hTF（并使用来自 hTF 的铁）；它甚至不绑定其他密切相关的 TF。影响识别的一些因素如下：无铁 hTF 和 LTF 结构的比较揭示了几个差异，这些差异对于这两种蛋白质的不同铁和受体结合特性可能很重要。虽然 hTF 和 LTF 在序列和结构上总体上非常相似，但它们的不同之处在于其叶间连接体的结构（LTF 中为螺旋状，hTF 中为非结构化）、LTF 的螺旋连接体与其 C 叶之间存在 hTF 中不存在的盐桥、它们的二硫键键合模式（hTF 中的亚结构域间键合，但在 LTF 中没有）、它们的叶的相对方向彼此之间的关系（与 hTF 相比，LTF 的 C 叶旋转得更靠近 N 叶）、hTF 中 LTF 中不存在的二赖氨酸触发剂和三联体残基、它们的 C 叶开放性（hTF 中更开放）、C 叶铰链区的结构（hTF 中非结构化，LTF 中 β 链）以及它们的叶间相互作用（hTF C 端螺旋和 N 叶之间的盐桥，在 LTF 中未发现）。对这些差异的分析增加了我们对这两种蛋白质不同功能的理解，以及病原菌表达两种相似蛋白质的独立受体的必要性。细菌受体与其宿主底物复合的晶体结构应该可以进一步了解这些相互作用。