Full-length Escherichia coli SecA dimerizes in a closed conformation in solution as determined by cryo-electron microscopy

Abstract

SecA is an obligatory component of the Escherichia coli general secretion pathway. However, the oligomeric structure of SecA and SecA conformational changes during translocation processes are still unclear. Here we obtained the three-dimensional structure of E. coli wild-type full-length SecA in solution by single particle cryo-electron microscopy and determined its oligomeric organization. In this structure, SecA occurs as a dimer in which the two protomers are arranged in an antiparallel mode, with a novel electrostatic interface, and both protomers are in closed conformation. The system developed here may provide a promising technique for studying dynamic structural changes in SecA.

FIGURE 1.

The E. coli SecA structure in solution. A, size-exclusion chromatography of SecA at 4 °C. A 125-μg sample of SecA was injected to a Superdex 200 column and eluted in a buffer of 20 mm HEPES-KOH, pH 7.5, or 20 mm HEPES-KOH, 300 mm KCl, pH 7.5. Marker proteins are: Ferritin (440 kDa), bovine catalase (232 kDa), and aldolase (158 kDa). B, cryo-EM image of 100 μg/ml SecA in buffer (20 mm Tris-Ac, pH 7.5, 1 mm DTT). Some individual particles are indicated by black arrows. The bar is 100 nm. C, resolution curves of the three-dimensional reconstruction. The resolutions calculated from two methods are shown: Fourier shell correlation (FSC) function (dashed line) and differential phase residual (DPR) function (solid line). The dotted line is the critical Fourier shell correlation function 3δ. D, distinct views of SecA cryo-EM samples. The top panel is the projection map of the reconstructed model; the bottom panel is the average map of all particles in this class; the number of particles in this class is indicated in the bottom panel. The box is 18.4 nm. E, surface representation of the three-dimensional reconstruction. The surface was rendered and displayed using the VMD software. Three views are shown: top view, side view, and bottom view. Each view was obtained after 90° rotation around the horizontal axis, as shown between the views. The three domains in the two subunits are designated as 1, 2, and 3, and 1′, 2′, and 3′.

FIGURE 2.

Docking of the cryo-EM three-dimensional map with the ecSecA crystal structure and comparison of the structures from cryo-EM and X-rays. A, chain A in the ecSecA (PDB number: 2FSF) was used for docking. The blue mesh represents the 1.5 σ contour of the electron density map. There are two molecules in the cryo-EM map, colored cyan and orange. Three views are shown. Each view is obtained after 90° rotation around the perpendicular or horizontal axis, as shown between the views. B, the docked crystal structure is rendered in different colors for different domains. For clarity, only one SecA protomer is shown. Similar to the assignment by Papanikolau et al. (19), ecSecA is divided into four parts: the NBD domain (purple); the Var, IRA2, and joint domains (yellow); the PBD domain (red); the C-terminal 30-kDa domain comprised of the IRA1, WD, SD, and CTD subdomains (green). As noted above, only a small fraction of PBD structure is seen in the refined x-ray structure. C, manual docking of one protomer (in different domain colors) in the x-ray dimeric structure (PDB number: 2FSF) into the electron density map. The colors in different domains were assigned as in B. The other protomer (orange) is obviously displaced in the EM map. The two side surfaces of the SecA protomer are designated Face A and Face B. D, view of Face A of the SecA protomer after electrostatic surface calculation. Electrostatic potential is represented as a tricolor gradient from blue (+1.8 kT (k, the constant of boltzmann; T, Thermodynamic temperature)) through white (neutral) to red (–5.8 kT), as represented using the Swiss-PDBviewer and calculated by DELPHY. E, view of Face B of the SecA protomer after electrostatic surface calculation. The patches of the C-domain, the IRA2 domain, and the NBD domain are indicated. The two protomers interact with each other through this interface. F and G, docking of the cryo-EM map with bsSecA x-ray structure in the closed (PDB number: 1M6N) (F) or open (PDB number: 1TF5) (G) state. For clarity, only one SecA protomer is shown. The PBD domain is red, and the C-domain is green. Both the side and the top view are shown.