Metal ion export in bacteria

描述（由申请人提供）：大肠杆菌等细菌已经开发出各种机制来克服不利于其生存的有毒环境。细菌用来破坏有毒化合物（包括重金属离子）的一个重要策略是表达膜转运蛋白，该膜转运蛋白识别并主动将这些有毒化合物排出细菌细胞，从而使细菌能够在极端有毒的条件下生存。我们的长期目标是阐明结构和基本机制，引起重金属离子识别和挤出的重金属外排蛋白。该提案的主要目标是E。coli CusABC外排系统，其识别并将银和铜离子挤出细菌细胞。CusA由1，047个氨基酸残基组成。它是一种内膜转运蛋白，属于抗性-增殖-分裂（RND）蛋白超家族。CusC是由457个氨基酸组成的多肽，在大肠杆菌中形成外膜通道。杆菌这两种膜蛋白相互作用，与膜融合蛋白CusB（379个氨基酸）结合，介导重金属离子穿过E.杆菌有人提出，CusB可能作为一个适配器，使CusA和CusC在一起，形成CusABC三方复合物。这种外排复合物与金属离子直接接触，并选择性地将它们排出细胞。我们最近克隆，表达和纯化的全长CusA，CusC和CusB外排蛋白。我们还用蒸汽扩散法使每种蛋白质在洗涤剂溶液中结晶.在同步辐射光源下从低温冷却的晶体收集X射线衍射数据。最好的CusA，CusC和CusB晶体衍射的分辨率分别为3.1，3.6和2.8 E，空间群确定为R32，P21和I222。具体的目的是确定重金属离子与以下物质相互作用的结构基础：（1）CusA内膜外排泵，（2）CusC外膜通道，和（3）CusB膜融合蛋白。

DESCRIPTION (provided by applicant): Bacteria such as Escherichia coli have developed various mechanisms to overcome toxic environments that are otherwise unfavorable for their survival. One important strategy that bacteria use to subvert toxic compounds, including heavy metal ions, is the expression of membrane transporters that recognize and actively export these toxic compounds out of bacterial cells, thereby allowing the bugs to survive in extremely toxic conditions. Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to heavy metal ion recognition and extrusion in heavy metal efflux proteins. The primary target of this proposal is the E. coli CusABC efflux system that recognizes and extrudes silver and copper ions out of the bacterial cell. CusA consists of 1,047 amino acid residues. It is an inner membrane transporter, which belongs to the resistance-nodulation-division (RND) protein superfamily. CusC is a 457 amino acid polypeptide that forms an outer membrane channel in E. coli. These two membrane proteins interact with each other, in conjunction with a membrane fusion protein CusB (379 amino acids), to mediate the extrusion of heavy metal ions across both membranes of E. coli. It has been proposed that CusB may act as an adaptor that brings CusA and CusC together to form the CusABC tripartite complex. This efflux complex makes direct contact with the metal ions and selectively expels them out of the cell. We recently cloned, expressed, and purified the full-length CusA, CusC, and CusB efflux proteins. We also crystallized each protein in detergent solution using vapor- diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best CusA, CusC and CusB crystals diffracted to resolutions of 3.1, 3.6, and 2.8 E, respectively, with space groups determined to be R32, P21, and I222. The specific aims are to determine the structural basis of heavy-metal ion interactions with: (1) the CusA inner membrane efflux pump, (2) the CusC outer membrane channel, and (3) the CusB membrane fusion protein.