Structure Determination Of Membrane Iron Transporters

膜铁转运蛋白的结构测定

• 批准号: 6820511
• 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/6820511
• 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 structure; protein structure function; receptor binding; siderophores; stoichiometry; structural biology; transferrin

Bacterial 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. Iron transport across the outer membrane is interesting to study because this family of transporters shows high affinity and specificity for Fe(III)-ligand complexes and because the transport process requires energy derived from the proton motive force across the inner membrane. The required energy is transduced by transient complexation with an integral inner membrane protein, TonB, resulting in a complex that spans both the inner and outer membranes, as well as the periplasmic space. The first crystal structure [1] of an E. coli TonB-dependent receptor, ferric enterobactin receptor (FepA = 80 kDa), revealed that this iron transporter uses a 22-stranded beta-barrel to span the outer membrane, with an unanticipated globular domain folded into the barrel interior. The globular domain appears to function in both ligand binding at the extracellular side of the membrane, and in TonB recognition at the periplasmic side of the membrane. In this 'ground state' structure, the globular domain inside the barrel completely occludes the barrel lumen, and we do not understand how ferric enterobactin (700 Da) is transported. To understand more about iron transport, three projects were initiated in May 2001. (1) Ligand recognition and transport Colicin I receptor (Cir) is a 67 kD Ton-B dependent outer membrane receptor which transports colicins Ia and Ib (~70 kDa) as well as its physiological iron-ligand. Since the structure of FepA does not indicate how a 700 Da molecule should pass through the beta-barrel, we are intrigued by the problem of transporting a protein that is 100 times larger. Colicin I receptor also functions in the uptake of catechol-substituted antibiotics and in the uptake of 2,3-dihydroxybenzoate derivatives. Particularly because colicin I receptor transports catechol-substituted antibiotics, a crystal structure will be useful to the field of antibiotic research. An added benefit from studying this particular receptor is that the structure of colicin Ia has been solved and the domain that should recognise and bind Cir has been identified. We have collected native data for Cir at 2.8A resolution and have introduced 4 additional methionine residues to aid phasing by the multiwavelength anomalous dispersion method. Structure solution is underway. We have also characterized the binding of colicin Ia to Cir by microcalorimetry. The complex has a nanomolar dissociation constant and co-crystallization experiments are in progress. (2) Regulation of iron transport at the E. coli outer membrane Escherichia coli K-12 synthesizes six siderophore-mediated transport systems for the acquisition of Fe3+ (such as colicin Ia and ferric enterobactin receptors, discussed above). Each system encodes a distinct transmembrane receptor for transport across the outer membrane. Transcription of ferric siderophore transport genes can be induced by extracellular ferric citrate. Induction involves FecA, the outer membrane transporter of ferric citrate, FecR, an inner membrane protein that transmits the signal into the cytoplasm, and FecI, a sigma factor that mediates specific binding of the RNA polymerase core enzyme to the promoter region upstream of fecA. We have just solved the structures of FecA alone and in complex with citrate and ferric citrate to resolutions of 2.1A to 3.4A [2]. These three structures together shed new light on apo- and holo-ligand binding. We have 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. The structure of FecA bound with iron-free dicitrate represents the first structure of a TonB-dependent transporter bound with an iron-free siderophore. Binding of diferric dicitrate to FecA results in changes in the orientation of the two citrate ions relative to each other and in their interactions with FecA, compared to the binding of iron-free dicitrate. The changes in ligand binding are accompanied by conformational changes in three areas of FecA: two extracellular loops, one plug domain loop and the periplasmic TonB-box motif. The positional and conformational changes in the siderophore and transporter initiate two independent events: ferric citrate transport into the periplasm and transcription induction of the fecABCDE transport genes. From these data we proposed a two-step ligand recognition event: FecA binds iron-free dicitrate in the non-productive state or first step, followed by siderophore displacement to form the transport-competent, diferric dicitrate bound state in the second step. (3) Structure of the Neisseria meningitidis transferrin binding protein complexed to human transferrin Neisseria meningitidis, a Gram-negative bacterium, is the causative agent of bacterial meningitidis. This blood-borne pathogen acquires iron from human transferrin (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. Both TbpA and TbpB are current vaccine targets. While TbpB is predicted to be a surface-exposed lipoprotein, TbpA is a member of the TonB-dependent outer membrane receptor/transporter family. We have determined the stoichiometry of the TbpA:TbpB:transferrin complex to be 2:1:1, yielding a total size of 360 kDa. Crystallisation experiments are underway for (a) TbpA alone, (b) TbpA in complex with transferrin, (c) TbpB alone, (d) TbpB in complex with transferrin, and (e) the entire TbpA:TbpB:tranferrin complex. Crystals have been obtained for TbpA complexed with the C-lobe of human transferrin, which diffract to 3.2A resolution. Additional methionine residues have been added to aid phasing. We expect that our crystal structures will aid vaccine development for serotype B meningitis, for which there is currently no vaccine available.

革兰氏阴性生物体中的细菌转运是由被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。跨外膜的铁转运值得研究，因为该转运蛋白家族对 Fe(III)-配体复合物表现出高亲和力和特异性，而且转运过程需要来自跨内膜的质子动力的能量。所需的能量通过与完整的内膜蛋白 TonB 的瞬时复合来转导，从而形成跨越内膜和外膜以及周质空间的复合物。 大肠杆菌 TonB 依赖性受体、铁肠杆菌素受体 (FepA ​​= 80 kDa) 的第一个晶体结构 [1] 揭示了这种铁转运蛋白使用 22 链 β 桶跨越外膜，并有一个意想不到的球状结构域折叠到桶内部。球状结构域似乎在膜胞外侧的配体结合和膜周质侧的 TonB 识别中发挥作用。在这种“基态”结构中，桶内的球状域完全遮挡了桶内腔，我们不了解肠杆菌素铁（700 Da）是如何运输的。为了更多地了解钢铁运输，2001 年 5 月启动了三个项目。 (1) 配体识别与运输 大肠菌素 I 受体 (Cir) 是一种 67 kD Ton-B 依赖性外膜受体，可转运大肠菌素 Ia 和 Ib (~70 kDa) 及其生理铁配体。由于 FepA ​​的结构并未表明 700 Da 分子应如何通过 β 桶，因此我们对运输 100 倍大的蛋白质的问题很感兴趣。大肠菌素 I 受体还在儿茶酚取代抗生素的摄取和 2,3-二羟基苯甲酸酯衍生物的摄取中发挥作用。特别是因为大肠菌素 I 受体转运儿茶酚取代的抗生素，晶体结构将有助于抗生素研究领域。研究这种特殊受体的另一个好处是，大肠菌素 Ia 的结构已经得到解决，并且识别和结合 Cir 的结构域也已经确定。我们以 2.8A 分辨率收集了 Cir 的原始数据，并引入了 4 个额外的蛋氨酸残基，以帮助通过多波长异常色散方法进行定相。结构解决方案正在进行中。我们还通过微量热法表征了大肠菌素 Ia 与 Cir 的结合。该复合物具有纳摩尔解离常数，共结晶实验正在进行中。 (2) 大肠杆菌外膜铁转运的调控 大肠杆菌 K-12 合成六种铁载体介导的转运系统来获取 Fe3+（例如上文讨论的大肠杆菌素 Ia 和铁肠杆菌素受体）。每个系统都编码一个独特的跨膜受体，用于跨外膜运输。细胞外柠檬酸铁可以诱导铁铁载体转运基因的转录。诱导涉及 FecA（柠檬酸铁的外膜转运蛋白）、FecR（一种将信号传递到细胞质中的内膜蛋白）和 FecI（一种介导 RNA 聚合酶核心酶与 fecA 上游启动子区域特异性结合的 Sigma 因子）。我们刚刚解析了单独的 FecA 以及与柠檬酸盐和柠檬酸铁的复合物的结构，分辨率为 2.1A 至 3.4A [2]。这三种结构共同为载脂蛋白和全息配体结合提供了新的线索。我们推断出区分无铁铁载体和三价铁铁载体的结构机制：二柠檬酸二铁的结合，而不是单独的无铁二柠檬酸，导致转运蛋白中的主要构象重排。与无铁二柠檬酸结合的 FecA 的结构代表了与无铁铁载体结合的 TonB 依赖性转运蛋白的第一个结构。与无铁的二柠檬酸的结合相比，二柠檬酸二铁与 FecA 的结合导致两个柠檬酸根离子相对于彼此的方向以及它们与 FecA 的相互作用发生变化。配体结合的变化伴随着 FecA 三个区域的构象变化：两个细胞外环、一个插头结构域环和周质 TonB-box 基序。铁载体和转运蛋白的位置和构象变化启动两个独立的事件：柠檬酸铁转运到周质中和fecABCDE转运基因的转录诱导。根据这些数据，我们提出了一个两步配体识别事件：FecA 在非生产状态或第一步结合无铁的二柠檬酸，然后在第二步中通过铁载体置换形成有运输能力的二柠檬酸二铁结合状态。 (3)与人转铁蛋白复合的脑膜炎奈瑟菌转铁蛋白结合蛋白的结构 脑膜炎奈瑟菌是一种革兰氏阴性细菌，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 (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 和 TbpB 都是当前的疫苗目标。 TbpB 预计是一种表面暴露的脂蛋白，而 TbpA 是 TonB 依赖性外膜受体/转运蛋白家族的成员。我们确定 TbpA:TbpB:转铁蛋白复合物的化学计量为 2:1:1，总大小为 360 kDa。 (a) 单独的 TbpA、(b) 与转铁蛋白复合的 TbpA、(c) 单独的 TbpB、(d) 与转铁蛋白复合的 TbpB 和 (e) 整个 TbpA:TbpB:转铁蛋白复合物的结晶实验正在进行中。已获得与人转铁蛋白 C 叶复合的 TbpA 晶体，其衍射分辨率为 3.2A。添加了额外的蛋氨酸残基以帮助定相。我们期望我们的晶体结构将有助于 B 血清型脑膜炎疫苗的开发，目前尚无疫苗可用。