Tol-dependent macromolecule import through the Escherichia coli cell envelope requires the presence of an exposed TolA binding motif - PubMed (original) (raw)

Tol-dependent macromolecule import through the Escherichia coli cell envelope requires the presence of an exposed TolA binding motif

Stéphanie Pommier et al. J Bacteriol. 2005 Nov.

Abstract

The Tol-Pal proteins of the cell envelope of Escherichia coli are required for maintaining outer membrane integrity. This system forms protein complexes in which TolA plays a central role by providing a bridge between the inner and outer membranes via its interaction with the Pal lipoprotein. The Tol proteins are parasitized by filamentous bacteriophages and group A colicins. The N-terminal domain of the Ff phage g3p protein and the translocation domains of colicins interact directly with TolA during the processes of import through the cell envelope. Recently, a four-amino-acid sequence in Pal has been shown to be involved in Pal's interaction with TolA. A similar motif is also present in the sequence of two TolA partners, g3p and colicin A. Here, a mutational study was conducted to define the function of these motifs in the binding activity and import process of TolA. The various domains were produced and exported to the bacterial periplasm, and their cellular effects were analyzed. Cells producing the g3p domain were tolerant to colicins and filamentous phages and had destabilized outer membranes, while g3p deleted of three residues in the motif was affected in TolA binding and had no effect on cell integrity or colicin or phage import. A conserved Tyr residue in the colicin A translocation domain was involved in TolA binding and colicin A import. Furthermore, in vivo and in vitro coprecipitation analyses demonstrated that colicin A and g3p N-terminal domains compete for binding to TolA.

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Figures

FIG. 1.

FIG. 1.

Amino acid sequences of the G3 and TA peptides. The recombinant colicin A translocation domain (TA) and g3pN1 (G3) are shown in panels A and B, respectively. The N-terminal OmpA signal sequences (lowercase italic letters), the Strep-tagged residues (capital and shaded boldface letters), TolA binding region of TA (31), and the residues of G3 in contact with TolA (41) are indicated (capital boldface letters) together with the first and last residues of the natural sequences of g3pN1 and colicin A translocation domain (capital and shaded letters). The mutations corresponding to residue deletions (capital and underlined boldface letters) and to Tyr-to-Ala mutations are shown (Tyr numbering is relative to the natural sequence of g3p and colicin A). The N-terminal residues of the translocation domain of colicin A, absent from the TA variants, containing the TolR binding sequence (lowercase letters) followed by a TolB box [consensus sequence: DG(T/S)G(S/W)] is shown in the panel A insert.

FIG. 2.

FIG. 2.

In vivo stability of TolA. (A) W3110 cells producing the indicated proteins were loaded on SDS-PAGE gels and transferred onto a nitrocellulose membrane. Immunoblots were revealed with anti-TolAII-III or anti-MalE antibodies or using Strep-tactin colorimetric detection for the TA and G3 variants. (B) W3110 cells were incubated in 10 mM potassium phosphate buffer (pH 7.1) in the absence or presence of colicin A or of the TR-truncated colicin A (TR-A, translocation and reception domain of colicin A) and harvested at the indicated times (min). About 2 × 108 cells were loaded on each lane. Molecular weight markers are indicated together with the degradation products of TolA (*). Ct, control empty vector.

FIG. 3.

FIG. 3.

In vitro interaction of TA and G3 variants with TolA. (A) Overlay immunodetections of TA and G3 variants bound to immobilized TolAIII. Serial twofold dilutions of TolAIII (3.0, 1.5, 0.8, 0.4, 0.2, and 0.1 μg for TA and 6.0, 3.0, 1.5, 0.8, 0.4, and 0.2 μg for G3) were spotted on a nitrocellulose membrane, incubated with the indicated TA or G3 variants, and further detected using Strep-tactin-coupled alkaline phosphatase. Ct indicates the TA or G3 proteins (1.0 μg for TA and 3.0 μg for G3) used for the control of colorimetric detection levels. (B) Gel shift analyses of TolAIII complexes. Purified TA, TA variants, ATh (the whole translocation domain of colicin A), and G3 were incubated with TolAIII and analyzed by Tricine-SDS-PAGE and Coomassie blue staining. (C) Copurifications of TolAII-III with TA or G3 variants. Copurifications of TolAII-III by Strep-tactin pull-down experiments of TA, G3, and variants were analyzed by Coomassie blue staining of SDS-PAGE (TA) or Tricine-SDS-PAGE (G3). Molecular weight markers are indicated.

FIG. 4.

FIG. 4.

TolA-TA complex formation in the presence of excess of Pal, g3pN1, or TolB. TA was incubated with the indicated proteins (concentrations, between 2 and 6 μM). Copurification of TolAII-III (A and C) or TolAIII (B) was assessed after TA pull-down assays using Strep-tag. When indicated, Pal (A), TolB (B), or g3pN1 (G3 devoid of Strep-tag) (C) was added to the mixture. An asterisk indicates that a 5-molar excess of the indicated protein was used. After SDS-PAGE (A and C) or Tricine-SDS-PAGE (B), proteins were detected by Coomassie blue staining. Molecular weight markers are indicated on the left.

FIG. 5.

FIG. 5.

Structures of TolAIII and its interacting partners. (A) Crystal structures of TolAIII (Protein Data Bank [PDB] accession number 1S62 [22]), Pal (PDB accession number 1OAP [C. Abergel, A. Walburger, E. Bouveret, and J.-M. Claverie, unpublished results]), and TolB (PDB accession number 1CRZ [1]). The SYGK motif of Pal, involved in its interaction with TolA, is modeled with balls and sticks. (B) Three-dimensional structure of the two N-terminal domains of g3p (PDB accession number 1G3P [40]). Residues of G3pN1 involved in the interaction with TolAIII but not participating in the interaction with G3pN2 (residues E14, NATG42, and GT56 of the YGT motif) and residues of G3pN2 involved in the interaction with the F-pilus are indicated by balls and sticks. (C) The YGT residues of g3pN1 involved in the contact with TolA are shown in the cocrystal structure of the g3pN1-TolAIII complex (PDB accession number 1TOL) together with Pal and its SYGK motif. The N-terminal membrane-anchored sequences of Pal and TolA are indicated by an asterisk. Figures were generated with 3D Mol Viewer (Vector NTI Suite version 8.0).

FIG. 6.

FIG. 6.

Sequence conservation of the TolA box. Sequences containing the TolA boxes of various TolA partners with their five flanking residues were aligned using TCoffee (

http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee\_cgi/index.cgi

). The positions of the N-terminal residues, the TolA box, and the conserved Tyr residue are indicated.

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References

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