Structural basis for the assembly of the Sec translocase machinery

Sec 易位机组装的结构基础

• 批准号: 8551289
• 负责人: CHARALAMPOS KALODIMOS
• 金额: $ 31.39万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8551289
• 关键词: ATP phosphohydrolase; Affect; Anti-Bacterial Agents; Bacteria; Bacterial Adhesins; Bacterial Proteins; Binding; Biochemical; Biological Assay; Complex; Cytosol; Data; Development; Docking; Escherichia coli; Hand; Human; Hydrolysis; Immune system; In Vitro; Kinetics; Membrane; Molecular Chaperones; Molecular Conformation; Motor; NMR Spectroscopy; Pathway interactions; Peptide Signal Sequences; Pharmaceutical Preparations; Play; Process; Protein translocation; Proteins; Resolution; Ribosomes; Role; Sampling; Scanning; Series; Structure; Thermodynamics; Toxin; Virulence; base; design; fascinate; in vivo; inhibitor/antagonist; insight; operation; periplasm; polypeptide; public health relevance; secretory protein; translocase

DESCRIPTION (provided by applicant): The Sec translocase is an astonishing nanomachinery responsible for translocating the vast majority of bacterial proteins destined for secretion from the cytosol to the periplasm or for integration into the membrane. Secretory proteins are synthesized with an N-terminally appended extension, the so-called signal sequence. As soon as the nascent chain of a newly synthesized protein starts emerging from the ribosome channel, it is typically greeted by the Trigger Factor (TF), the only chaperone that associates with the ribosome in bacteria. TF is thought to scan the nascent polypeptide chain and protect it from aggregation. Secretory proteins are targeted to the Sec pathway and through a series of discrete steps they are finally threaded through the translocon channel. The secretory protein first interacts with the SecB chaperone, whose role is to retain the protein substrate in an unfolded, translocation-competent conformation. Then, the protein substrate is handed-over to SecA, an ATPase motor protein. In the final step, the SecA-protein substrate complex docks onto the SecYEG membranous translocon channel and through successive cycles of ATP binding to SecA and hydrolysis the protein substrate is translocated through the channel. Thus, central to the functionality of the translocase is the timely and coordinated action of several proteins: the TF chaperone, the SecB chaperone, the SecA ATPase, and the SecYEG translocon channel. Despite the significant progress made over the past two decades in understanding the mode of operation of this multi-protein machinery, fundamental questions about the functional and structural mechanisms underpinning its assembly and operation remain unaddressed. More specifically, there is very little known about how the unfolded secretory protein is recognized and bound by TF and the Sec proteins and how the protein substrate is handed over from one protein to the other as the process moves downstream from the ribosome to the translocon. We propose to use an integrated approach combining structural, dynamic, thermodynamic, kinetic, biochemical and in vitro and in vivo functional assays to provide insight into all of the steps involving the interaction of the secretory protein substrate with TF, SecB an SecA as well as the formation of binary and ternary complexes required for ultimately targeting the protein at the translocon. We have extensively characterized over the last years all of the protein components from Escherichia coli and have obtained and present here strong data supporting intriguing emerging hypotheses about the mechanisms used by the Sec machinery to carry out its function. The specific aims are designed to provide atomic-resolution insight into (i) the mechanisms of interaction between the protein substrate and the TF chaperone, (ii) the mechanisms controlling the entry of the secretory protein substrate into the Sec pathway, (iii) the holdase chaperone activity of SecB, and (iv) the targeting of the protein substrate to the SecA ATPase.

描述（由申请人提供）：Sec 转位酶是一种令人惊叹的纳米机器，负责将绝大多数细菌蛋白转位，这些蛋白将从细胞质分泌到周质或整合到膜中。分泌蛋白的合成具有 N 末端附加的延伸，即所谓的信号序列。一旦新合成的蛋白质的新生链开始从核糖体通道中出现，它通常会受到触发因子（TF）的欢迎，触发因子是细菌中与核糖体相关的唯一伴侣。 TF被认为可以扫描新生的多肽链并保护其免于聚集。分泌蛋白靶向 Sec 途径，并通过一系列离散步骤，最终穿过易位子通道。分泌蛋白首先与 SecB 伴侣相互作用，其作用是将蛋白质底物保留在未折叠的、易位的构象中。然后，蛋白质底物被移交给 SecA（一种 ATP 酶马达蛋白）。在最后一步中，SecA-蛋白质底物复合物对接至 SecYEG 膜易位通道，并通过 ATP 与 SecA 结合和水解的连续循环，蛋白质底物通过通道易位。因此，易位酶功能的核心是及时和协调的行动 几种蛋白质的组成：TF 伴侣、SecB 伴侣、SecA ATP 酶和 SecYEG 易位子通道。尽管过去二十年来在理解这种多蛋白机器的运作模式方面取得了重大进展，但有关支撑其组装和运作的功能和结构机制的基本问题仍未得到解决。更具体地说，关于未折叠的分泌蛋白如何被 TF 和 Sec 蛋白识别和结合，以及当该过程从核糖体向下游移动到易位子时，蛋白底物如何从一种蛋白转移到另一种蛋白，人们知之甚少。我们建议使用一种结合结构、动力学、热力学、动力学、生物化学以及体外和体内功能测定的综合方法，以深入了解涉及分泌蛋白底物与 TF、SecB 和 SecA 相互作用的所有步骤，以及最终将蛋白质靶向易位子所需的二元和三元复合物的形成。在过去的几年里，我们对大肠杆菌的所有蛋白质成分进行了广泛的表征，并获得并在此展示了强有力的数据，支持关于 Sec 机器执行其功能的机制的有趣的新假设。具体目标旨在提供原子分辨率的洞察力 (i) 蛋白质底物和 TF 伴侣之间的相互作用机制，(ii) 控制分泌蛋白质底物进入 Sec 途径的机制，(iii) SecB 的保持酶伴侣活性，以及​​ (iv) 蛋白质底物靶向 SecA ATP 酶。