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

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.

Use of synthetic signal sequences to explore the protein export machinery

“…Supporting this was our finding using biotin-labeled LamB signal peptides that signal peptides bind more strongly to C-terminally truncated SecA64, which has properties similar to the open translocation-active state, than to full-length SecA. 72 This observation supports the existence of two binding sites: a low-affinity binding site in cytosolic SecA and a high-affinity site in the activated form. IRA1 may be acting as a molecular switch to regulate signal sequence binding, 67 since its deletion also increased binding.…”

“…69,72 These results support an interdependent role of IRA1 and signal peptides during the translocation cycle. 69,89 A System to Explore How Signal Peptides Bind the Open Conformation of SecA…”

“…Early work by several laboratories including our own showed that disruption or removal of the C-terminal domain of SecA led to elevation of its ATPase activity to levels near to those associated with translocation, increased signal sequence binding, and dissociation of the other domains. 72,74,75,88 In parallel, this C-terminal region was shown to be the region inserted into the membrane as SecA became a membrane-resident protein. 75,76,79,80 Thus, the picture that emerges is that the ligand-mediated triggering of domain dissociation leads to insertion of SecA into the membrane and activation of its translocase function.…”

“…Studies from a variety of groups over the past decade have revealed that SecA is a multidomain protein that can exist as a monomer or a dimer, and that its domain rearrangements can be triggered by binding to any of its several ligands. 67,[69][70][71][72][73][74][75][76][77][78][79][80][81] The key to the conformational rearrangements appears to be intramolecular interactions that stabilize a more tightly packed, ''closed'' form of the protein. Weakening these interactions relieves these intramolecular constraints, and the protein domains partially dissociate, creating a less tightly packed, ''open'' form of the protein.…”

The ATPase domain of SecA can form a tetramer in solution 1 1Edited by I. B. Holland

“…36 Finally, the N-terminal domain appears to be largely responsible for SecYEG binding. 37 The C-terminal domain is important for mediating dimer formation between SecA protomers; 38,39 it has an important role in regulating the ATPase activity and ligand-binding properties of the N-terminal domain, 32,36,39 and is also required for binding to SecB. 40 We are interested in the mechanisms by which SecA couples ATP binding and hydrolysis to interactions with preprotein and SecYEG.…”