The Escherichia coli SecA ATPase motor protein is essential for secretion of proteins through the SecYEG translocon into the periplasmic space. Its function relies upon interactions with the surrounding lipid bilayer as well as SecYEG translocon. That negatively charged lipids are required for bilayer binding has been known for >25 years, but little systematic quantitative data is available. We have carried out an extensive investigation of SecA partitioning into large unilamellar vesicles (LUV) using a wide range of lipid and electrolyte compositions, including the principal cytoplasmic salt of E. coli, potassium glutamate, which we have shown stabilizes SecA. The water-to-bilayer transfer free energy is about -7.5 kcal mol^-1 for typical E. coli lipid compositions. Although it has been established that SecA is dimeric in the cytoplasm, we find that the most widely cited dimer form (PDB 1M6N) binds only weakly to LUVs formed from E. coli lipids.

The essential SecA motor ATPase acts in concert with the SecYEG translocon to secrete proteins into the periplasmic space of Escherichia coli. In aqueous solutions, SecA exists largely as dimers, but the oligomeric state on membranes is less certain. Crystallographic studies have suggested several possible solution dimeric states, but its oligomeric state when bound to membranes directly or indirectly via the translocon is controversial. We have shown using disulfide crosslinking that the principal solution dimer, corresponding to a crystallographic dimer (PDB 1M6N), binds only weakly to large unilamellar vesicles (LUV) formed from E. coli lipids. We report here that other soluble crosslinked crystallographic dimers also bind weakly, if at all, to LUV. Furthermore, using a simple glutaraldehyde crosslinking scheme, we show that SecA is always monomeric when bound to LUV formed from E. coli lipids.

The soluble cytoplasmic ATPase motor protein SecA powers protein transport across the Escherichia coli inner membrane via the SecYEG translocon. Although dimeric in solution, SecA associates monomerically with SecYEG during secretion according to several crystallographic and cryo-EM structural studies. The steps SecA follows from its dimeric cytoplasmic state to its active SecYEG monomeric state are largely unknown. We have previously shown that dimeric SecA in solution dissociates into monomers upon electrostatic binding to negatively charged lipid vesicles formed from E. coli lipids. Here we address the question of the disposition of SecA on the membrane prior to binding to membrane embedded SecYEG. We mutated to cysteine, one at a time, 25 surface-exposed residues of a Cys-free SecA. To each of these we covalently linked the polarity-sensitive fluorophore NBD whose intensity and fluorescence wavelength-shift change upon vesicle binding report on the the local membrane polarity. We established from these measurements the disposition of SecA bound to the membrane in the absence of SecYEG. Our results confirmed that SecA is anchored in the membrane interface primarily by the positive charges of the N terminus domain. But we found that a region of the nucleotide binding domain II is also important for binding. Both domains are rich in positively charged residues, consistent with electrostatic interactions playing the major role in membrane binding. Selective replacement of positively charged residues in these domains with alanine resulted in weaker binding to the membrane, which allowed us to quantitate the relative importance of the domains in stabilizing SecA on membranes. Fluorescence quenchers inside the vesicles had little effect on NBD fluorescence, indicating that SecA does not penetrate significantly across the membrane. Overall, the topology of SecA on the membrane is consistent with the conformation of SecA observed in crystallographic and cryo-EM structures of SecA-SecYEG complexes, suggesting that SecA can switch between the membrane-associated and the translocon-associated states without significant changes in conformation.

Much is known about the structure, function, and stability of the SecA motor ATPase that powers the secretion of periplasmic proteins across the inner membrane of Escherichia coli. Most studies of SecA are carried out in buffered sodium or potassium chloride salt solutions. However, the principal intracellular salt of E. coli is potassium glutamate (KGlu), which is known to stabilize folded proteins and protein-nucleic acid complexes. Here we report that KGlu stabilizes SecA, including its dimeric state, and increases its ATPase activity, suggesting that SecA is likely fully folded, stable, and active in vivo at 37°C. Furthermore, KGlu also stabilizes a precursor form of the secreted maltose-binding protein.