Metal ion export in bacteria

描述(申请人提供)：大肠杆菌等细菌已经开发出各种机制来克服有毒环境，否则这些环境不利于它们的生存。细菌用来颠覆包括重金属离子在内的有毒化合物的一个重要策略是表达膜转运蛋白，该转运蛋白识别并主动将这些有毒化合物输出到细菌细胞外，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在重金属外排蛋白中引起重金属离子识别和排泄的结构和基本机制。这项提议的主要目标是大肠杆菌CusABC外排系统，该系统识别并将银离子和铜离子排出细菌细胞。CUSA由1047个氨基酸残基组成。它是一种内膜转运蛋白，属于抗性结瘤分裂(RND)蛋白超家族。CusC是一种由457个氨基酸组成的多肽，在大肠杆菌中形成外膜通道。这两个膜蛋白与膜融合蛋白CusB(379个氨基酸)相互作用，介导重金属离子穿过大肠杆菌的两个膜。有观点认为，CusB可能作为一个接头，将CUSA和CusC连接在一起，形成CusABC三方复合体。这种外排复合体与金属离子直接接触，并选择性地将它们驱逐出细胞。我们最近克隆、表达和纯化了CusA、CusC和CusB外排蛋白的全长。我们还用气相扩散法在洗涤剂溶液中结晶了每种蛋白质。X射线衍射数据是在同步加速器光源下从低温晶体中收集的。CusA、CusC和CusB晶体的衍射率分别为3.1、3.6和2.8E，空间群分别为R32、P21和I222。具体目的是确定重金属离子与：(1)CusA内膜外排泵，(2)CusC外膜通道，(3)CusB膜融合蛋白相互作用的结构基础。 与公共卫生相关：对重金属产生耐药性的病原体似乎正在上升。这一建议涉及跨膜外排转运体中Ag+/Cu+识别和挤出的结构基础。因此，这项研究将有助于提高我们对病原菌对银和铜的耐药性的了解，并将为思考新的基于金属的抗菌治疗策略提供一个平台，从而导致新的治疗方法。

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. PUBLIC HEALTH RELEVANCE: Heavy-metal resistance pathogens appear to be on the rise. This proposal deals with the structural basis of Ag+/Cu+ recognition and extrusion in transmembrane efflux transporters. Thus, the research will help to improve our knowledge of silver and copper resistance in pathogenic bacteria, and will provide a platform for thinking about novel metal-based antimicrobial therapeutic strategies that will lead to new treatments.