Synthetic peptides identify a second periplasmic site for the plug of the SecYEG protein translocation complex

A short helix in the centre of the SecY subunit serves as a 'plug' blocking the protein channel. This site must be vacated if the channel is to open and accommodate translocating protein. We have synthesised a peptide mimic of this plug, and show that it binds to E. coli SecYEG, identifying a distinct and peripheral binding site. We propose that during active translocation the plug moves to this second discrete site and chart its position. Deletion of the plug in SecY increases the stoichiometry of the peptide-SecYEG interaction by also exposing the location it occupies in the channel. Binding of the plug peptide to the channel is unaffected by SecA.