Alignment of the protein substrate hairpin along the SecA two-helix finger primes protein transport in Escherichia coli.

A conserved hairpin-like structure comprised of a signal peptide and early mature region initiates protein transport across the SecY or Sec61α channel in Bacteria or Archaea and Eukarya, respectively. When and how this initiator substrate hairpin forms remains a mystery. Here, we have used the bacterial SecA ATPase motor protein and SecYEG channel complex to address this question. Engineering of a functional miniprotein substrate onto the end of SecA allowed us to efficiently form ternary complexes with SecYEG for spectroscopic studies. Förster resonance energy transfer mapping of key residues within this ternary complex demonstrates that the protein substrate adopts a hairpin-like structure immediately adjacent to the SecA two-helix finger subdomain before channel entry. Comparison of ADP and ATP-γS-bound states shows that the signal peptide partially inserts into the SecY channel in the latter state. Our study defines a unique preinsertion intermediate state where the SecA two-helix finger appears to play a role in both templating the substrate hairpin at the channel entrance and promoting its subsequent ATP-dependent insertion.