Binding of the motor protein SecA to the bacterial protein translocation channel SecYEG in the absence of ATP

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by the protein translocation complex SecYEG. The motor protein SecA is thought to use ATP hydrolysis to push the polypeptide chain through SecYEG. How SecA may perform several rounds of ATP hydrolysis is unclear, since its nucleotide-free conformation was reported to be unable of forming stable complexes with SecYEG in detergent. In contrast, we now observed long lasting SecA binding events to the reconstituted SecYEG complex by high-speed atomic force microscopy. Their duration suggests an apparent affinity in the order of 10 nM. Between two consecutive binding events, the nucleotide-free SecA remained attached to neighboring lipids. To confirm the hypothesis that the high affinity is due to the concerted action of two low affinity sites on SecA, we employed (i) surface plasmon resonance for the characterization of SecAs binding to lipid bilayers and (ii) luminescence resonance energy transfer for the investigation of SecAs binding to SecYEG. Energy transfer between a genetically engineered Tb3+ binding pocket on reconstituted SecY and a fluorescent dye on the tip of SecAs two-helix finger reported two conformational states: one with a deep and one with a rather shallow protrusion of the two helix finger into the SecYEG channel. The two states correspond well to two different classes of ion channel activity observed by single channel recordings. In turn, channel lifetimes were consistent with long and short binding events that we visualized by high speed atomic force microscopy. Equilibrium constants of (i) ~ 60 μM for the interactions of the two helix finger with pore lining residues of SecY and (ii) ~0.2 μM for the binding of SecAs N-terminus to lipids may act in unison to ensure SecAs capability to reside on SecYEG between two consecutive ATP binding events.