Regulatory and recognition mechanisms of translocase ATPase SecA

描述（由申请人提供）：SecA是细菌Sec转位酶机制中高度保守和必需的解旋酶样马达蛋白。SecA识别分泌蛋白，并将其通过跨膜SecYEG通道的运输与ATP结合和水解提供的代谢能量的消耗结合起来。我们的长期目标是：(i)阐明SecA解旋酶马达的调控特性，并了解它们在ATP催化循环中是如何被控制的；（ii）描述使SecA能够区分分泌蛋白和非分泌蛋白的识别机制；（iii）提供SecA与其所有易位配体相互作用的结构基础。提出的研究背后的假设是，SecA (i)在激活过程中切换到高度灵活的状态，其中无序-有序转换调节电机的特性，以及（ii）通过使用交替结合位点来完成混杂信号序列识别。基于初步观察，具体目标旨在提供以下原子分辨率的见解：(i)在催化循环中SecA如何在功能构象状态之间切换，（ii）信号序列混杂结合的识别机制，以及（iii） SecA与前蛋白底物，SecB伴侣和SecYEG的相互作用。我们的目标是：1；描述在atp酶周期中SecA马达的构象和动态特性，以了解马达功能的潜在机制。2. 阐明由SecA识别混杂信号肽的分子决定因素，以了解单个转位酶系统如何能够识别和分泌多种蛋白质底物。3. 通过核磁共振表征SecA与其易位伙伴（前蛋白，SecB, SecYEG）的相互作用，以了解级联结合现象如何导致完整易位机制的组装。细菌感染后，SecA负责数百种蛋白质底物的分泌，其中包括几种影响人体免疫系统的毒素和粘附素。该提案的长期目标是为开发针对SecA的抗菌抑制剂提供适当的物理化学基础。

DESCRIPTION (provided by applicant): SecA is a highly conserved and essential helicase-like motor protein of the bacterial Sec translocase machinery. SecA recognizes secretory proteins and couples their transport through the transmembrane SecYEG channel with the expenditure of metabolic energy provided by ATP binding and hydrolysis. Our long term goals are to (i) elucidate the regulatory properties of the helicase motor of SecA and understand how they are controlled during the ATP catalytic cycle, (ii) delineate the recognition mechanisms that enable SecA to differentiate between secretory and non-secretory proteins, and (iii) provide the structural basis of the interaction of SecA with all of its translocation ligands. The hypothesis behind the proposed research is that SecA (i) switches to a highly flexible state during its activation, wherein disorder-order transitions regulate the properties of the motor, and (ii) accomplishes the promiscuous signal sequence recognition by using alternate binding sites. Based on preliminary observations, the specific aims are designed to provide atomic-resolution insight into (i) how SecA switches among functional conformational states during the catalytic cycle, (ii) the recognition mechanisms of the promiscuous binding of signal sequences, and (iii) the interaction of SecA with preprotein substrates, the SecB chaperone and SecYEG. We aim to: 1. Delineate the conformational and dynamic properties of SecA's motor during the ATPase cycle, to understand the underlying mechanisms of the motor functionality. 2. Elucidate the molecular determinants of the promiscuous signal peptide recognition by SecA, to understand how a single translocase system is able to recognize and secrete a wide variety of protein substrates. 3. Characterize by NMR the interaction of SecA with its translocation partners (preprotein, SecB, SecYEG), to understand how the cascade of the binding phenomena results in the assembly of the complete translocase machinery. Upon bacterial infection, SecA becomes responsible for the secretion of hundreds of protein substrates, among them several toxins and adhesins that affect the human immune system. A long-term goal of the proposal is to provide the appropriate physicochemical basis towards development of antibacterial inhibitors targeting SecA.