SecA functions in vivo as a discrete anti‐parallel dimer to promote protein transport

Abstract

Summary SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer-dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ρ-benzoylphenylalanine along dimer interfaces corresponding to the five different SecA x-ray structures and assessing their in vivo photo-crosslinking pattern. A discrete anti-parallel 1M6N-like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG-bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer-driven translocation mechanism.

The SecA protein deeply penetrates into the SecYEG channel during insertion, contacting most channel transmembrane helices and periplasmic regions

“…see P100 or K434 in Fig. 3A) consistent with earlier studies (37,40,41). This result arises because of the broad chemical reactivity of pBpA and its ability to cross-link to two or more neighboring residues of the target to create products of differing axial radii when analyzed by SDS-PAGE.…”

Substrate Proteins Take Shape at an Improved Bacterial Translocon

“…Finally, a third model utilizes both the conformational changes of the dynamic SecA monomer-dimer cycle and the reciprocating action of the THF to drive SecA-bound substrate into the channel in two successive steps (the reciprocating piston model) (34,43). Indeed, SecA monomer-dimer dynamics (44) appear to play a more proximal role during the substrate protein handoff step from SecB to SecA, since association of the N terminus of SecA with the C terminus of SecB would dissociate the 1M6N-like SecA homodimer, which has been shown to be the relevant form in vivo (45,46). Later on, the SecA N terminus also contributes to SecYEG targeting through its amphipathic anionic phospholipid-binding activity, as discussed above.…”

An alternate mode of oligomerization for E. coli SecA

“…Residue 15, the first residue present in the SecA-N68∆NC structure, is indicated with a red sphere. Sites of in vivo photo-activated cross-linking are indicated by residues highlighted with spheres and coloured cyan for one study 26 and blue for a second study 25 . ( E ) The antiparallel dimer viewed down its two-fold rotation axis (top panel) and rotated 90° about a vertical axis; in this case the N-terminal amino acids of E. coli SecA from residue 3 (indicated by “NT”) onwards, were modelled from the B. subtilis structure.…”

“…The parallel SAXS-based dimer incorporates the same interface as the anti-parallel crystallographic dimer, and on this basis is also consistent with much of the previous work characterizing the structure of the solution dimer. This includes in vivo photo-activated cross-linking studies 25 , 26 : cross-linking sites identified in these studies are highlighted in the dimer structures in Fig. 6 .…”

“…In the case of the general secretory system of E. coli , the available evidence does not provide a conclusive answer as to the number of SecA subunits required for translocation. Attempts to address this question have been confounded by the fact that SecA exists as a dimer in solution 21 – 24 , but given the changes in SecA oligomerization required for its interaction with SecYEG, it is not clear whether the solution dimer is related to oligomeric species that may participate in the translocation reaction 13 , 25 . It is conceivable that interactions between SecA protomers could function to regulate ATPase activity as well as binding between SecA and the preprotein during translocation.…”