Functional signal peptides bind a soluble n-terminal fragment of SecA and inhibit its ATPase activity

The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is kn own about the requirements for recognition and the mode of binding of precu rsors to SecA, The major reason for this is the lack of a soluble system su itable for biophysical study of the SecA-precursor complex. Complicating th e development of such a system is the likelihood that SecA interacts with t he precursor in a high affinity, productive manner only when it is activate d by binding to membrane and SecYEG. A critical aspect of the precursor/Sec A 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 func tions of SecA can be studied using separated domains. Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, w hich is stably folded, has high ATPase activity, and represents an activate d 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 exten t that correlates with the ability of these signal peptides to inhibit eith er SecA translocation ATPase or in vitro protein translocation, arguing tha t the interaction with SecA64 is functionally significant. Thus, SecA64 off ers a soluble, well defined system to study the mode of recognition of sign al peptides by SecA and the regulation of signal peptide release.