Structure Determination Of Bacterial Outer Membrane Prot

革兰氏阴性菌的转运是由转运的物种通过外膜中的跨膜β-桶启动的。铁的运输对细菌的生长特别重要，因此外膜铁转运体是对抗奈瑟氏菌、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)-配体络合物表现出高度的亲和力和特异性，并且需要来自穿过内膜的质子动力的能量来运输铁络合物。所需的能量是通过与完整的内膜蛋白复合体TonB-ExbB-ExbD的瞬时相互作用提供的，导致蛋白质组装跨越内膜和外膜以及周质空间。在2006财年，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。这种血液传播的病原体通过外膜转运蛋白结合蛋白A和B(TbpA=100 kDa；TbpB=68-85 kDa)从人转铁蛋白(HTF=80 kDa)中获取铁。TbpA和TbpB形成一个离散的复合体，协同结合转铁蛋白，但每个蛋白也能单独结合转铁蛋白。TbpA是一种依赖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。 我们分别表达和纯化了TbpA和TbpB，为研究TbpA-TbpB-HTF三重复合体做准备。这项工作在前几年的年度报告中进行了总结。为了实现我们确定三重复合体晶体结构的目标，我们首先需要解决人血清转铁蛋白的结构，这是一种30多年来一直无法确定结构的蛋白质。 血清转铁蛋白可逆地结合两个叶的铁，并通过受体介导的、pH依赖的过程将其输送到细胞。铁的结合和释放导致了较大的构象变化，其中每个叶中的两个亚域通过绕铰链的刚性扭曲运动关闭或打开。用两种方法独立测定了缺铁人血清转铁蛋白(apo-HTF)的结构：(1)重组非糖化apo-HTF的晶体结构在2.7？(2)糖基化apo-HTF(从血清中分离)的结构测定结果为2.7？以人的载脂蛋白N-叶和兔的HALO-C_1亚区为搜索模型进行分子置换。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转铁蛋白的模型，并揭示了与家族成员(人乳铁蛋白和母鸡卵转铁蛋白)相比，两个叶几乎是同样开放的：62？52岁呢？分别需要旋转才能打开N形和C形波瓣。这种结构的可用性对于完全理解HTF每个叶的金属结合性质是至关重要的；apo-HTF结构表明N-叶和C-叶的铰链区的差异可能会影响铁的结合和释放的速率。 一旦获得了全长HTF结构(参考文献3在发表列表中)，我们就能够对HTF和人乳铁蛋白进行结构比较(参考文献4在发表列表中)。关于TbpA如何区分HTF和乳铁蛋白的问题出现了，因为这两种蛋白质有很高的序列相似性，并且以相似的方式结合铁。然而，TpbA只结合(和使用)HTF中的铁；它甚至不结合其他密切相关的TF。影响识别的一些因素如下：比较无铁HTF和LTF的结构，揭示了这两种蛋白质不同的铁和受体结合特性的几个重要区别。尽管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：它们的叶间连接体的结构(在LTF中是螺旋的，在HTF中是非结构化的)，在LTF的螺旋连接体和它的C叶之间存在盐桥，它们的二硫键模式(在HTF中没有亚区间键合，但在LTF中不存在)，它们的叶彼此的相对取向(与HTF相比，LTF的C叶旋转得更接近N叶)，在HTF中不存在的双赖氨酸触发器和三联体残基，C叶的开放(在HTF中更加开放)，C叶铰链区的结构(在HTF中是非结构化的)和##946；-链)，及其叶间相互作用(HTF的C-末端螺旋和N-叶之间的盐桥，这在LTF中没有)。对这些差异的分析有助于我们了解这两种蛋白质的不同功能，以及病原菌表达两种类似蛋白质的独立受体的必要性。细菌受体与其宿主底物的复合体的晶体结构应该提供对这些相互作用的进一步了解。

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.