The Escherichia coli outer membrane cobalamin transporter BtuB:: Structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation
The Escherichia coli outer membrane cobalamin transporter BtuB:: Structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation
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DOI：
10.1016/j.jmb.2003.07.005
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发表时间：
2003-10-03
影响因子：
5.6
通讯作者：
Wiener, MC
中科院分区：
文献类型：
--
作者：
Chimento, DP;Kadner, RJ;Wiener, MC
革兰氏阴性细菌具有专门的主动转运系统，其功能是将有机金属辅因子或载体，如钴离子、铁载体和卟啉，穿过它们的外膜。每个转运系统的主要组成部分是外膜转运蛋白和能量偶联蛋白TonB。在大肠杆菌中，TonB依赖性外膜转运蛋白BtuB进行钴胺素（Cbl）底物跨其外膜的主动转运。钴以纳摩尔亲和力结合BtuB。先前的研究涉及氰钴胺（CN-Cbl）与BtuB的高亲和力结合中的钙。我们以前解决了四种结构的BtuB或BtuB复合物：甲硫氨酸取代突变体的载脂蛋白结构（用于通过硒代甲硫氨酸单波长异常衍射研究获得实验阶段）;野生型BtuB的载脂蛋白结构;钙和野生型BtuB的二元复合物;和钙，CN-Cbl和野生型BtuB的三元复合物。我们提出了一个分析的二元和三元复合物中的钙的结合，并表明，钙配位底物结合后的变化。高亲和力CN-Cbl结合和钙配位是偶联的。我们还分析了CN-Cbl与BtuB的结合模式，并将这种结合与结合Cbl的其他蛋白质中观察到的结合进行比较和对比。BtuB结合CN-Cbl的方式非常不同的Cbl利用酶和周质Cbl结合蛋白BtuE细菌基因组的同源性搜索，结构注释的基础上的存在下，通过分析我们的BtuB结构确定的保守的Cbl结合残基，和检测的周质Cbl结合蛋白BtuF的同源物，使推定的BtuB直系同源物在肠道和非肠道细菌物种的鉴定。(C)2003爱思唯尔有限公司。保留所有权利。
Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes. The primary components of each transport system are an outer membrane transporter and the energy-coupling protein TonB In Escherichia coli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane. Cobalamins bind to BtuB with nanomolar affinity. Previous studies implicated calcium in high-affinity binding of cyanocobalamin (CN-Cbl) to BtuB. We previously solved four structures of BtuB or BtuB complexes: an apo-structure of a methionine-substitution mutant (used to obtain experimental phases by selenomethionine single-wavelength anomalous diffraction studies); an apo-structure of wild-type BtuB; a binary complex of calcium and wild-type BtuB; and a ternary complex of calcium, CN-Cbl and wildtype BtuB. We present an analysis of the binding of calcium in the binary and ternary complexes, and show that calcium coordination changes upon substrate binding. High-affinity CN-Cbl binding and calcium coordination are coupled. We also analyze the binding mode of CN-Cbl to BtuB, and compare and contrast this binding to that observed in other proteins that bind Cbl. BtuB binds CN-Cbl in a manner very different from Cbl-utilizing enzymes and the periplasmic Cbl binding protein BtuE Homology searches of bacterial genomes, structural annotation based on the presence of conserved Cbl-binding residues identified by analysis of our BtuB structure, and detection of homologs of the periplasmic Cbl-binding binding protein BtuF enable identification of putative BtuB orthologs in enteric and nonenteric bacterial species. (C) 2003 Elsevier Ltd. All rights reserved.