Functional Signal Peptides Bind a Soluble N-terminal Fragment of SecA and Inhibit Its ATPase Activity

Terry L Triplett,Anita R Sgrignoli,Fen-Biao Gao,Yun-Bor Yang,Phang C Tai,Lila M Gierasch

doi:10.1074/jbc.m100098200

Abstract

The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is known about the requirements for recognition and the mode of binding of precursors to SecA. The major reason for this is the lack of a soluble system suitable for biophysical study of the SecA-precursor complex. Complicating the development of such a system is the likelihood that SecA interacts with the precursor in a high affinity, productive manner only when it is activated by binding to membrane and SecYEG. A critical aspect of the precursor/SecA interaction is that it is regulated by various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the release of the precursor, most likely in a stepwise fashion, for translocation. Several recent reports show that functions of SecA can be studied using separated domains. Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated version of SecA. We report here that this fragment, termed SecA64, binds signal peptides with significantly higher affinity than does SecA. Moreover, the ATPase activity of SecA64 is inhibited by signal peptides to an extent that correlates with the ability of these signal peptides to inhibit either SecA translocation ATPase or in vitro protein translocation, arguing that the interaction with SecA64 is functionally significant. Thus, SecA64 offers a soluble, well defined system to study the mode of recognition of signal peptides by SecA and the regulation of signal peptide release.

Highlights

SecA plays a central role in bacterial protein export: recognition of the protein precursor and facilitation of its transloca
To ensure the physiological relevance of the interactions between SecA64 and synthetic signal peptides, we have examined the effects of several of the same signal peptides on translocation ATPase and in vitro protein translocation, both of which were previously shown to be inhibited by the addition of signal peptides [13,14,15]
Removal of approximately one-third of the SecA molecule by limited chymotrypsinolysis provided us with SecA64, a soluble, activated 64-kDa form of this large molecule with which to examine its function as a signal sequence receptor

Summary

Introduction

SecA plays a central role in bacterial protein export: recognition of the protein precursor and facilitation of its transloca-. Since SecA64 has properties suggesting that it resembles the activated state of SecA critical for pre-protein delivery to the membrane, we were interested in determining how signal sequence binding modulates its functions. We have used peptides corresponding to wild-type and mutant signal sequences from the Escherichia coli outer membrane proteins, LamB and OmpA, to explore signal sequence binding to SecA64.

Results

Conclusion