STRUCTURES OF SIDEROPHORE MEMBRANE TRANSPORT PROTEINS

铁载体膜运输蛋白的结构

• 批准号: 2910053
• 负责人: DICK VAN DER HELM
• 金额: $ 16.6万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910053
• 关键词: Escherichia coli; X ray crystallography; acid base balance; aluminum; bacterial proteins; calcium; gallium; indium; iron; membrane permeability; membrane transport proteins; metal complex; point mutation; protein structure function; receptor; receptor binding; siderophores

Many aerobic microorganisms produce siderophores which are secreted into the environment. The siderophores chelate Fe3+ specifically. The complexation constants are very high (10\22-10/50). The complexes are too large (M.W. 800-1500) to be transported by porins. For competitive reasons microorganisms produce often more than one siderophore, and the structures of approximately 100 siderophores are known. Also, for better competition, bacteria produce more than one uptake system for ferric siderophores. For instance, under iron deficient conditions, E. coli has an uptake system for ferric enterobactin, the siderophore E. coli produces, consisting of FepA (in the outer membrane), FepB, C, D, and G in the periplasm and inner membrane. However, E. coli also has an uptake system for ferrichrome which it does not produce. This uptake system uses FhuA in the outer membrane and associated Fhu proteins in the periplasm and inner membrane. E. coli also has a citrate inducible uptake system for ferric citrate which uses FecA in the O.M. It is our plan, in cooperation with Dr. Deisenhofer, to determine the crystal structures of FepA, FhuA and their complexes with Fe enterobactin and ferrichrome, respectively, as well as FecA. All these proteins have homology but are different from any known protein. Native data sets (2.8 angstroms) have been taken for FepA, diffraction-quality crystals are available for Se-Met-FepA and both FepA-Fe(ent) and FhuA-ferrichrome complexes have been crystallized. The proteins require detergents also for crystallization. The O.M. of gram negative bacteria has no energy source. TonB, anchored in the I.M., is known to interact with FepA and FhuA. We hypothesize in this proposal a detailed mechanism for transport which can be probed by the structure determinations and the planned binding experiments which are described. The specificity of transport resides in the O.M. proteins, FepA and FhuA. This specificity will be probed: a) by point mutations in the protein (primarily FepA); b) by using structurally similar siderophores (primarily for FhuA); and c) by changing the Fe3+ in the complexes for similar ions (GA3+, Al3+ and In3+). This will be done with binding experiments and difference Fouriers. The intent of the proposal is to understand, in detail, the specific transport of large molecules across a membrane.

许多有氧微生物产生的铁载体被分泌到 环境。 铁载体螯合Fe3+。 这 络合常数非常高（10 \ 22-10/50）。 复合物是 太大（M.W. 800-1500）将被波林运输。 具有竞争力 原因是微生物通常会产生一个以上的辅助载体，而 已知大约100个铁载体的结构。 此外，为了更好地竞争，细菌产生多种吸收 铁铁载体系统。 例如，在铁不足的情况下 条件，大肠杆菌具有摄取摄取系统的摄取系统， 辅助大肠杆菌产生，由FEPA组成（在外部 膜），FEPB，C，D和G中的膜和内膜。 但是，大肠杆菌也有一个用于铁晶的吸收系统 不产生。 该吸收系统在外膜中使用FHUA， 在周围和内膜中相关的FHU蛋白。 大肠杆菌 还具有用于使用柠檬酸铁的柠檬酸盐诱导摄取系统 O.M. 这是我们的计划，与Deisenhofer博士合作 确定FEPA，FHUA及其复合物的晶体结构 分别与Fe Enterobactin和Ferrichrome一起以及FECA。 所有这些蛋白质都有同源性，但与任何已知的 蛋白质。 为FEPA采用了天然数据集（2.8埃） 衍射质量晶体可用于SE-MET-FEPA，两者都可以 FEPA-FE（ENT）和FHUA-FERRICHROME复合物已结晶。 这 蛋白质还需要洗涤剂才能结晶。 O.M.革兰氏阴性细菌没有能源。 Tonb，锚定 在I.M.中，已知与FEPA和FHUA相互作用。 我们假设 在此提案中，可以探讨的详细运输机制 通过结构确定和计划的结合实验 描述的。 运输的特异性位于O.M. 蛋白质，FEPA和FHUA。 将探测此特异性：a） 蛋白质中的突变（主要是FEPA）； b）通过结构上使用 类似的铁载体（主要用于FHUA）； c）通过更改FE3+ 在类似离子的复合物中（Ga3+，Al3+和In3+）。 这将是 使用结合实验和差异傅立叶。 意图 该提议是要详细了解 跨膜的大分子。