Structural dynamics of peptide-translocating ABC transporters

蛋白质跨膜输出是生命的关键过程。在原核生物中， 运输系统的发展促进了两亲性和亲水性蛋白质的转移 令人印象深刻的大小跨越脂质疏水屏障。这些系统中最简单的包括 ATP结合盒（ABC）转运蛋白，其利用ATP水解的能量， 为货物肽或蛋白质穿过细胞膜的运动提供动力。革兰阳性 细菌，ABC转运蛋白输出抗微生物或群体感应肽， 赋予生物体在有限资源条件下的生存优势。长 本申请的长期目标是阐明含肽酶的多肽的构象动力学， ABC转运蛋白（PCAT），它利用一个内置的半胱氨酸蛋白酶结构域来切割信号 在输出之前，货物肽的序列。我们战略的前提是：1） PCAT的最新高分辨率结构，定义了它们的分子结构， 详细研究这些转运蛋白使蛋白质 易位和2）PI在最先进的电子应用中的跟踪记录 顺磁共振（EPR）光谱结合致突变分析， 运输机具体目标旨在解决该领域尚未解决的问题 包括由ATP周转驱动的交替访问的结构基础， 货物蛋白的特异性，以及货物蛋白的结构和环境，因为它 通过传送器传送在过去的十年里，我们的方法与 分子模拟在揭示多药物的结构动力学方面是有用的 ABC出口商，并提供深入了解这个家庭内的机械多样性。的 这项研究的重要性源于一个分子靶点， 抗菌肽转运，细菌免疫，代表了一个基本的生化 蛋白质通过脂质双层输出的过程。

Protein export across membranes is a critical process for life. In prokaryotes, multiple protein transport systems were evolved to facilitate the transfer of amphipathic and hydrophilic proteins of impressive sizes across lipid hydrophobic barriers. The simplest of these systems consist of an ATP binding Cassette (ABC) transporter which harnesses the energy of ATP hydrolysis to power the movement of cargo peptides or proteins across the cell membranes. In gram positive bacteria, ABC transporters export antimicrobial or quorum sensing peptides which serve to endow the organism with survival advantages under conditions of limited resources. The long term goal of this application is to illuminate the conformational dynamics of peptidase-containing ABC transporters (PCATs) which utilizes a built in cysteine protease domain to cleave the signal sequence of the cargo peptide prior to export. The premise of our strategy is grounded in 1) recent high resolution structures of PCATs that define their molecular architecture and set the stage for detailed investigation of the mechanism by which these transporters enable protein translocation and 2) the track record of the PI in the application of state of the art electron paramagnetic resonance (EPR) spectroscopy in conjunction with mutagenic analysis to active transporters. The specific aims are designed to address unanswered questions in the field including the structural basis of alternating access powered by ATP turnover, the determinants of cargo protein specificity, and the structure and environment of the cargo protein as it transitions through the transporter. Over the last decade, our approach integrated with molecular modeling has been instrumental in revealing the structural dynamics of multidrug ABC exporters and providing insight into the mechanistic diversity within this family. The significance of the proposed research stems from a molecular target at the junction of antibacterial peptide transport, bacterial immunity and represents a fundamental biochemical process by which proteins are exported across lipid bilayers.