Structural basis for the assembly of the Sec translocase machinery

描述(申请人提供)：SEC转位酶是一种惊人的纳米机械，负责将绝大多数细菌蛋白质从胞浆分泌到周质或整合到膜中。分泌蛋白的合成带有N-末端的延伸，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，它通常就会受到触发因子(Tf)的欢迎，这是细菌中唯一与核糖体相关的伴侣。转移因子被认为扫描新生的多肽链并保护其免受聚集。分泌蛋白以SEC途径为靶点，通过一系列离散的步骤，最终通过转位蛋白通道。分泌蛋白首先与SecB伴侣相互作用，SecB伴侣的作用是将蛋白质底物保持在未折叠的、可转位的构象中。然后，蛋白质底物被移交给一种ATPase马达蛋白SecA。在最后一步中，SecA-蛋白质底物复合体对接到SecYEG膜转位蛋白通道上，通过与SecA结合和水解的连续循环，蛋白质底物通过该通道转运。因此，转位酶功能的核心是及时和协调的行动 有几种蛋白质：转铁蛋白伴侣、SecB伴侣、SecA ATPase和SecYEG转位蛋白通道。尽管在过去20年里，在理解这种多蛋白质机制的运作模式方面取得了重大进展，但关于支撑其组装和运作的功能和结构机制的基本问题仍然没有得到解决。更具体地说，关于未折叠的分泌蛋白是如何被TF和SEC蛋白识别和结合的，以及当这个过程从核糖体下游转移到转运子时，蛋白质底物是如何从一个蛋白质传递到另一个蛋白质的，人们知之甚少。我们建议使用一种综合的方法，结合结构、动力学、热力学、动力学、生化和体外和体内功能分析来深入了解分泌蛋白底物与Tf、SecB和SecA相互作用的所有步骤，以及最终将蛋白质靶向转位蛋白所需的二元和三元复合体的形成。在过去的几年里，我们已经广泛地表征了来自大肠杆菌的所有蛋白质成分，并在这里获得并展示了强有力的数据，支持关于SEC机制用于执行其功能的机制的有趣的新兴假说。特定的目标旨在提供原子分辨率的洞察 (I)蛋白质底物与转铁蛋白伴侣相互作用的机制，(Ii)控制分泌蛋白底物进入SEC途径的机制，(Iii)SecB的保持酶伴侣活性，(Iv)蛋白质底物对SecA-ATPase的靶向。

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.