Defining the Escherichia coli SecA dimer interface residues through in vivo site-specific photo-cross-linking

Abstract

The motor protein SecA is a core component of the bacterial general secretory (Sec) pathway and is essential for cell viability. Despite evidence showing that SecA exists in a dynamic monomer-dimer equilibrium favoring the dimeric form in solution and in the cytoplasm, there is considerable debate as to the quaternary structural organization of the SecA dimer. Here, a site-directed photo-cross-linking technique was utilized to identify residues on the Escherichia coli SecA (ecSecA) dimer interface in the cytosol of intact cells. The feasibility of this method was demonstrated with residue Leu6, which is essential for ecSecA dimerization based on our analytical ultracentrifugation studies of SecA L6A and shown to form the cross-linked SecA dimer in vivo with p-benzoyl-phenylalanine (pBpa) substituted at position 6. Subsequently, the amino terminus (residues 2 to 11) in the nucleotide binding domain (NBD), Phe263 in the preprotein binding domain (PBD), and Tyr794 and Arg805 in the intramolecular regulator of the ATPase 1 domain (IRA1) were identified to be involved in ecSecA dimerization. Furthermore, the incorporation of pBpa at position 805 did not form a cross-linked dimer in the SecA Δ2-11 context, indicating the possibility that the amino terminus may directly contact Arg805 or that the deletion of residues 2 to 11 alters the topology of the naturally occurring ecSecA dimer.

Fig 1

Sedimentation velocity analysis of SecA (A) and SecA Δ2-11 (B) in 300 mM KCl buffer, shown as normalized g(s*) distributions. Each distribution represents a different protein concentration. The distributions are normalized by amplitude. The sedimentation velocity measurements were performed as described in Materials and Methods.

Fig 2

Expression, identification, and specificity of SecA site-specific in vivo photo-cross-linking. (A) SDS-PAGE analysis of the induction of wild-type SecA (WT) and SecA L6X, S402X, and Y820X in the absence (−) and in the presence (+) of pBpa. Full-length SecA is produced only in the presence of pBpa. The arrowheads indicate SecA fragments that likely prematurely terminated at the amber codon. Their molecular masses are consistent with the location of the amber codon used for pBpa incorporation. The corresponding fragment from SecA L6X is so small that it cannot be detected by 7.5% SDS-PAGE. (B) SDS-PAGE analysis of SecA L6X expression at the indicated times after IPTG induction. Un, uninduced cells. (C) Western blot analysis of lysates from either untreated (−) or UV-irradiated (+) cells expressing SecA L6X harvested at the indicated times following induction. SecA dimer and SecA–EF-Tu, confirmed by LC-MS/MS, and monomeric SecA are indicated. (D) Comparison of the UV cross-linking pattern of SecA L6X and those of wild-type SecA (WT) and SecA T340X. Samples for panels A and B were from whole-cell lysates. Samples for panels C and D were from the soluble fraction of cell lysates. A monoclonal anti-His antibody was used for immunoblot analysis as described in Materials and Methods. Lanes M, protein molecular mass markers, with molecular masses shown on the left.

Fig 3

SecA residues 2 to 11 are involved in SecA dimerization in vivo. Lysates from UV-treated (+) cells expressing pBpa-containing SecA (A) and pBpa-containing SecA and SecA-c-myc (B) were analyzed via Western blotting and probed with antibodies specific to His tag (A) and to c-myc tag (B). The corresponding nontreated (−) cells are also shown. The amount of sample proteins loaded for panel B is 3 times higher than that for panel A. To reduce the interference from the monomer bands, the gels in panel B were run longer to allow the monomer to migrate out of the gel (same in Fig. 4B). Cross-linked SecA dimer bands are indicated with asterisks. Lanes M, protein molecular mass markers, with molecular masses shown on the left.

Fig 4

Phe263, Tyr794, and Arg805 are found on the SecA dimer interface. (A) Residues investigated by in vivo photo-cross-linking are labeled on the ecSecA nuclear magnetic resonance (NMR) structure (PDB ID, 2VDA) with the signal peptide masked. The structure presentation is colored according to SecA domain organization as follows: NBD, light blue; IRA2, pale cyan; PBD, light pink; SD, violet; WD, gray; IRA1, pale yellow; and CTD, orange. The colored spheres show the positions of the amber mutations. Black spheres indicate the variants expressed as insoluble proteins, and green, blue, and red spheres indicate the variants yielding no, weak, and strong cross-linked SecA dimer, respectively. Residues showing strong cross-linking signals are labeled. (B) Cross-linked SecA F263X, Y794X, and R805X dimer bands were confirmed by Western blotting with anti-His and anti-c-myc antibodies. (C) No cross-linked dimer bands form when the flanking residues of Phe263, Tyr794, or Arg805 are replaced with pBpa and UV irradiated. Cross-linked dimer bands are indicated with asterisks. Lanes M, protein molecular mass markers, with molecular masses shown on the left.

Fig 5

Comparison of cross-linking of variants F263X, Y794X, and R805X on the background of SecA and SecA Δ2-11 after 1 h and 2 h of induction. Cross-linked dimer bands are indicated with asterisks. Lane M, protein molecular mass markers, with molecular masses shown on the left.

Fig 6

Positions of the ecSecA cross-linked residues identified here are overlaid on different SecA dimer crystal structures. One protomer (light green) is shown in cartoon and transparent surface representations and the other (gray-blue) in cartoon form only. The dimer interface revealed by the crystal structures is in yellow. Phe263, Tyr794, and Arg805 of ecSecA (2FSF-PBD) and their equivalent residues as defined by the sequence alignment program ClustalW on mtSecA (Tyr251, Met798, and Arg809 on 1NL3), bsSecA (Phe250, Tyr743, and Met754 on 2IBM or Div_dimer), and ttSecA (Tyr264, Tyr901, and Ile912 on 2IPC) are labeled and shown in red single spheres. To denote the location of the amino terminus, residue 10 in each structure is shown by four purple spheres, except in 2IBM, where it is residue 11 of chain B (gray-blue).