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.

许多需氧微生物产生铁载体，这些铁载体被分泌到 环境。 铁载体专门螯合 Fe3+。 这 络合常数非常高 (10\22-10/50)。 这些复合物是 太大（M.W. 800-1500）而无法通过孔蛋白运输。 为了有竞争力 微生物经常产生多个铁载体的原因，以及 已知大约 100 个铁载体的结构。 此外，为了更好的竞争，细菌会产生不止一种吸收 三价铁铁载体系统。 例如，缺铁时 条件下，大肠杆菌具有铁肠杆菌素的摄取系统， 大肠杆菌产生铁载体，由 FepA（在外层）组成 膜），周质和内膜中的 FepB、C、D 和 G。 然而，大肠杆菌也有一个铁铬摄取系统，它确实 不生产。 该摄取系统在外膜中使用 FhuA， 周质和内膜中的相关 Fhu 蛋白。 大肠杆菌 还具有柠檬酸铁的柠檬酸盐诱导吸收系统，该系统使用 O.M. 中的 FecA 这是我们与 Deisenhofer 博士合作的计划， 确定 FepA、FhuA 及其复合物的晶体结构 分别含有 Fe 肠杆菌素和铁铬，以及 FecA。 所有这些蛋白质都具有同源性，但与任何已知的蛋白质不同 蛋白质。 FepA ​​采用本机数据集（2.8 埃）， 衍射质量晶体可用于 Se-Met-FepA ​​以及两者 FepA-Fe(ent) 和 FhuA-铁铬复合物已结晶。 这 蛋白质也需要去垢剂才能结晶。 O.M.的革兰氏阴性细菌没有能量来源。 TonB，锚定 在 I.M. 中，已知与 FepA ​​和 FhuA 相互作用。 我们假设 在这个提案中，有一个可以探索的详细传输机制 通过结构测定和计划的结合实验 所描述的。 运输的特殊性在于 O.M. 蛋白质、FepA ​​和 FhuA。 将通过以下方式探究这种特异性：a) 逐点 蛋白质突变（主要是 FepA）； b) 通过使用结构 类似的铁载体（主要针对 FhuA）； c) 通过改变 Fe3+ 在相似离子（GA3+、Al3+ 和 In3+）的配合物中。 这将是 通过结合实验和差分傅立叶完成。 的意图 该提案旨在详细了解 跨膜的大分子。