The SecA ATPase motor protein binds to Escherichia coli liposomes only as monomers

Abstract

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.

Keywords: Disulfide crosslinking; Glutaraldehyde crosslinking; Large unilamellar vesicles (LUV); Protein partitioning; Protein secretion.

Figure 1.

SecA does not bind as one of the crystallographic dimers to large unilamellar vesicles (LUV) formed from Escherichia coli lipids. (A) Three-dimensional structures of six reported crystallographic dimers. The N-terminus of SecA, which is necessary for membrane binding [10, 50], is highlighted in red in one of the protomers. The top panel corresponds to the dimer species that are unlikely to bind to the membrane without major steric clashes with the membrane. In support of this hypothesis, we note that an earlier study we showed that the 1M6N dimer cannot bind significantly to E. coli LUV [10]. The bottom panel shows the dimer species that potentially could exist on the lipid bilayer without steric clashes with the bilayer. (B) Coomassie-blue stained SDS-PAGE denaturing gel of cysteine-mutants of SecA (1 μM) in the absence of LUVs under oxidizing conditions. Note the presence of both monomers and dimers for 3JV2, 2FSF, and 1NL3 but not 1M6N. (C) Coomassie-blue stained SDS-PAGE gel of cysteine-mutants of SecA (1 μM) in the presence of LUVs (E. coli lipids, 6 mM). Partitioning of SecA was allowed for 30 minutes prior to introducing the oxidizer. In this case, no dimers are observed.

Figure 2.

Non-specific cross-linking of WT-SecA using glutaraldehyde (GA). (A) Side view of monomeric Escherichia coli SecA (PDB: 2FSF) with the membrane-partitioning N-terminus colored in red and lysine residues that are potential glutaraldehyde cross-linking sites highlighted in cyan. (B) Coomassie-blue stained SDS-PAGE gel of SecA protein (1 μM) in solution in the absence or presence of 0.15% GA. SecA in solution in the absence of vesicles was exposed to GA for 15 secs before halting the reaction with Tris-HCl. (C) GA-crosslinked SecA dimers do not partition significantly into E. coli LUV. The black curve shows the partitioning of untreated SecA (data from [49]). Titration of GA-crosslinked SecA (1 μM; 0.15% GA for 15 seconds) with LUV monitored by the change in intrinsic fluorescence is shown by the red curve. F₀ is the fluorescence intensity at 340 nm in the absence of lipids, and F is the intensity in the presence of vesicles.

Figure 3.

Dimeric SecA dissociates into monomers upon binding to LUV. (A) In the presence of LUV, there is a progressive shift from monomers to dimers with crosslinking time. After binding to 6 mM LUV (POPC:POPG:CL=0.7:0.2:0.1) for 30 minutes at 37°C, GA was introduced (0.15%) and allowed to crosslink for the periods shown before quenching the reaction. We conclude that as the cross-linking time increases, there is a shift from membrane-bound monomers to unbound dimers in solution. (B) Coomassie-blue stained SDS-PAGE denaturing gels showing the results of titrating SecA (1 μM) with lipid vesicles made of POPC (grey), POPC:POPG (0.7:0.3, black), or POPC:CL (0.7:0.3, red). In each case, partitioning was allowed for 30 minutes at 37°C before introducing GA (0.15%) for 15 seconds before stopping the reaction by the addition of an excess of 100 mM Tris-HCl pH 7.0). Note the progressive shift from dimers to monomers with increases in lipid concentration. (C) The changes in relative intensities of the bands on the gel as the lipid concentration is increased allows one to compute the fraction of SecA partitioned into the LUV at a given lipid concentration. The fraction partitioned f_P is given by [M]/([M] + [D]) where [M] is given by the intensity of the monomer band and [D] is the intensity of dimer band. The color code is the same as panel B. The water-to-bilayer free energies of transfer ΔG_wb for LUV formed from POPC:POPG and POPC:CL as −7.0 ± 0.3 and −7.1 ± 0.2 kcal.mol⁻¹, respectively. These values agree well with the value of ΔG_wb determined by fluorescence measurements of SecA partitioning into LUV formed from E. coli lipids (−7.4 ± 0.1 kcal mol⁻¹) [10]