Dimeric SecA couples the preprotein translocation in an asymmetric manner

Abstract

The Sec translocase mediates the post-translational translocation of a number of preproteins through the inner membrane in bacteria. In the initiatory translocation step, SecB targets the preprotein to the translocase by specific interaction with its receptor SecA. The latter is the ATPase of Sec translocase which mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. We examined the structures of Escherichia coli Sec intermediates in solution as visualized by negatively stained electron microscopy in order to probe the oligomeric states of SecA during this process. The symmetric interaction pattern between the SecA dimer and SecB becomes asymmetric in the presence of proOmpA, and one of the SecA protomers predominantly binds to SecB/proOmpA. Our results suggest that during preprotein translocation, the two SecA protomers are different in structure and may play different roles.

Figure 1. Complex formation between SecA and SecB.

(A) Absorbance profiles of SecA and SecB complexes resolved by size-exclusion chromatography. Red line: SecA alone (6 µM monomer). Green line: SecB alone (40 µM monomer). Cyan line: SecA monomer (3 µM) and SecB monomer (30 µM). Marker protein: aldolase (150 kDa), catalase (232 kDa), and ferritin (440 kDa). The inserted SDS-PAGE panel shows the contents for one fraction (from 10.0 to 10.3 mL) of the cyan line after TCA precipitation with Coomassie Blue staining. (B) EM image of negatively stained SecA/SecB complex. Distinct particles are indicated by arrows. The scale bar represents 50 nm. (C) Surface representation of the 3D reconstruction of the SecA/SecB complex (C1, C2, C3, C4). The two SecA protomers were rendered with different colors: green and red. The three domains in each SecA protomer are designated 1, 2, and 3, and 1′, 2′, and 3′. Four views are shown: side (C1, C2, C3) and top (C4). Each view was obtained after a 90° rotation operation around the axis as shown between these views. (D) Docking of the SecB (PDB ID: 1QYN) and closed E. coli SecA X-ray crystal structures (ecSecA-closed) into the EM 3D map. The three views are the same views as the C2–C4 respectively. The X-ray crystal structure of each SecA is colored green or red. The X-ray crystal structure of SecB is rendered in yellow. The contour EM density map of the SecA/SecB complex is represented by grey hatch markers. The scale bars represent 5 nm in (C) and (D).

Figure 2. Complex formation of SecA, SecB and proOmpA.

(A) Absorbance profiles of complexes of SecA, SecB, and proOmpA resolved by size-exclusion chromatography. The samples applied were 3 µM SecA monomer (red) and a mixture of 3 µM SecA monomer and 2 µM SecB/proOmpA (black). Fractions (300 µL) were collected and analyzed by SDS-PAGE with Coomassie Blue staining. The insert panel shows the result for the fraction with the highest absorbance at 280 nm. (B) Electron microscopy image of negatively stained SecA/proOmpA/SecB complexes. Some distinct particles are indicated by arrows. The scale bar represents 50 nm. (C) Surface representation of the 3D reconstruction of the SecA/proOmpA/SecB complex (C1, C2, C3, C4). The two SecA protomers are rendered in green and red. The unstained domain represents SecB/proOmpA. The three domains in each SecA protomer are designated 1, 2, and 3, and 1′, 2′, and 3′. Four views are shown of the surface representation: side view (C1, C2, C3) and top view (C4). Each view was obtained after the rotation operation around the axis as shown between these views. (D) Docking of the E. coli SecA (ecSecA-open) and SecB (PDB: 1QYN) X-ray structures into the EM 3D map. The three views are the same views as the C2–C4 respectively. The X-ray crystal structure of each E. coli SecA is colored green or red. The X-ray crystal structure of SecB is rendered in yellow. The contour of the EM density map of the SecA/proOmpA/SecB complex is represented with grey hatch markers. The scale bars represent 5 nm in (C) and (D).