STRUCTURE OF A MEMBRANE TRANSPORTER WITH INHIBITOR

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 CLC氯转运蛋白协调氯的运动，是正常的神经元、肌肉、心血管和上皮功能所必需的。确定了大肠杆菌CLC(ClC-EC1)与Fab片段形成的复合体的结构(PdB 10OTS)。在国家卫生研究院(1R01GM070773-01A2)资助的工作中，我们发现了一种ClC-EC1的抑制剂DIDS，它的表观亲和力为~30um。在这项工作中，我们建议确定CLC-EC1/DIDS复合体的结构。这些实验的结果将提供CLC抑制剂结合位点的第一个结构。这种结构将有助于使用DIDS作为工具来探索ClC-EC1中的氯运输机制，并可能被证明是有用的定位在DIDS抑制的真核同源物中的DIDS结合部位。此外，这种结构可能有助于设计更高亲和力的抑制剂，而这是CLCs严重缺乏的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The CLC chloride-transport proteins orchestrate the movement of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. The structure of an E. coli CLC (ClC-ec1) in complex with a Fab fragment has been determined (pdb 1OTS). In work funded by the NIH (1R01GM070773-01A2), we have discovered an inhibitor of CLC-ec1, DIDS, which has an apparent affinity of ~30 uM. In this work, we propose to determine the structure of the ClC-ec1/DIDS complex. The results of these experiments will provide the first structure of a CLC inhibitor binding site. This structure will facilitate the use of DIDS as a tool to probe the chloride-transport mechanism in CLC-ec1, and could also prove useful for mapping the DIDS-binding site in the eukaryotic homologs that are inhibited by DIDS. In addition, the structure may aid in designing higher-affinity inhibitors, which are sorely lacking for the CLCs.