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.
The Sec translocase is responsible for secreting hundreds of protein substrates, among them several toxins and adhesins that affect the human immune system. Optimization and development of antibacterial inhibitors can be used as potent drugs against virulence bacteria.
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