Regulatory and recognition mechanisms of translocase ATPase SecA

描述（由申请人提供）：SecA是细菌Sec易位酶机制的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通道的转运与ATP结合和水解提供的代谢能量的消耗偶联。我们的长期目标是（i）阐明SecA的解旋酶马达的调节特性，并了解它们在ATP催化循环过程中是如何控制的，（ii）描绘识别机制，使SecA能够区分分泌和非分泌蛋白，以及（iii）提供SecA与其所有易位配体相互作用的结构基础。这项研究背后的假设是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）通过使用替代结合位点完成混杂信号序列识别。基于初步的观察，具体的目的是提供原子分辨率的洞察（i）SecA如何在催化循环过程中的功能构象状态之间切换，（ii）信号序列的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SecA马达在ATP酶循环过程中的构象和动力学特性，以了解马达功能的潜在机制。2.阐明SecA识别混杂信号肽的分子决定因素，以了解单个移位酶系统如何能够识别和分泌各种蛋白质底物。3.通过NMR表征SecA与其易位伴侣（前蛋白，SecB，SecYEG）的相互作用，以了解结合现象的级联如何导致完整易位酶机制的组装。在细菌感染后，SecA负责分泌数百种蛋白质底物，其中包括影响人类免疫系统的几种毒素和粘附素。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.