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The crystal structure of the TolB box of colicin A in complex with TolB reveals important differences in the recruitment of the common TolB translocation portal used by group A colicins.

Zhang Y, Li C, Vankemmelbeke MN, Bardelang P, Paoli M, Penfold CN, James R - Mol. Microbiol. (2009)

Bottom Line: Comparison of this structure with that of the colicin E9 (ColE9) TolB box-TolB complex, together with site-directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB.Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA.This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection, Immunity and Inflammation, School of Molecular Medical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Interaction of the TolB box of Group A colicins with the TolB protein in the periplasm of Escherichia coli cells promotes transport of the cytotoxic domain of the colicin across the cell envelope. The crystal structure of a complex between a 107-residue peptide (TA(1-107)) of the translocation domain of colicin A (ColA) and TolB identified the TolB box as a 12-residue peptide that folded into a distorted hairpin within a central canyon of the beta-propeller domain of TolB. Comparison of this structure with that of the colicin E9 (ColE9) TolB box-TolB complex, together with site-directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB. Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA. This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.

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ColA binds to TolB but does not competitively recruit TolB from a TolB–Pal complex. A. Analytical gel filtration showing the individual protein peaks attributed to TolB, TE91-61::DNase and TE91-61::DNase–TolB complex. B. Analytical gel filtration showing the individual protein peaks attributed to TolB, TA1–107 and TA1–107–TolB complex. C. Analytical gel filtration showing the protein peaks attributed to Pal, TE91-61::DNase, the TolB–Pal and TE91-61::DNase–TolB complexes, and the peaks produced from a mixture of TolB–Pal incubated stoichiometrically with TE91-61::DNase that shows the displacement of Pal (peak 3) as TolB is competitively recruited by TE91-61::DNase (peak 1). Residual TolB–Pal (peak 2) remains due to incomplete recruitment of TolB in vitro. D. In contrast when TolB and Pal were mixed together, incubated stoichiometrically with TA1–107, and run on gel filtration the absence of a protein peak with the same retention time as TA1–107–TolB demonstrates no competitive recruitment of TolB by TA1–107. Protein peaks attributed to Pal, TA1–107, TolB–Pal and TA1–107–TolB are shown.
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fig05: ColA binds to TolB but does not competitively recruit TolB from a TolB–Pal complex. A. Analytical gel filtration showing the individual protein peaks attributed to TolB, TE91-61::DNase and TE91-61::DNase–TolB complex. B. Analytical gel filtration showing the individual protein peaks attributed to TolB, TA1–107 and TA1–107–TolB complex. C. Analytical gel filtration showing the protein peaks attributed to Pal, TE91-61::DNase, the TolB–Pal and TE91-61::DNase–TolB complexes, and the peaks produced from a mixture of TolB–Pal incubated stoichiometrically with TE91-61::DNase that shows the displacement of Pal (peak 3) as TolB is competitively recruited by TE91-61::DNase (peak 1). Residual TolB–Pal (peak 2) remains due to incomplete recruitment of TolB in vitro. D. In contrast when TolB and Pal were mixed together, incubated stoichiometrically with TA1–107, and run on gel filtration the absence of a protein peak with the same retention time as TA1–107–TolB demonstrates no competitive recruitment of TolB by TA1–107. Protein peaks attributed to Pal, TA1–107, TolB–Pal and TA1–107–TolB are shown.

Mentions: It has recently been shown that the binding surfaces of ColE9 and Pal with TolB are identical and that, in the presence of Ca2+, the TolB box of ColE9 competitively recruits TolB from Pal, presumably as a means of destabilizing the outer membrane on route to cell killing (Loftus et al., 2006). The Kd value of 1.6 µM for binding of TA1–107 to TolB in the presence of Ca2+ is much higher than the value of 90 nM reported for the binding of Pal to TolB in the presence of Ca2+ (Loftus et al., 2006), and thus makes it unlikely that the TolB box of ColA will competitively recruit TolB from the TolB–Pal complex. We confirmed this prediction by using analytical gel filtration chromatography. An interaction was observed between TolB and TE91-61::DNase (Fig. 5A), which is a chimeric polypeptide consisting of the first 61 residues of ColE9 fused to the DNase domain (Macdonald et al., 2004), or TA1–107 (Fig. 5B). However, when the pre-formed TolB–Pal complex was incubated, in the presence of Ca2+, with an equivalent concentration of TE91-61::DNase (Fig. 5C), or TA1–107 (Fig. 5D), only the ColE9 NDR was able to competitively recruit TolB as shown by a retention peak (peak 1) that overlapped the retention peak produced by TE91-61::DNase–TolB, and a retention peak (peak 3) of free Pal (Fig. 5C). As TE91-61::DNase–TolB and TolB–Pal have affinities of binding of 84 nM and 90 nM respectively, in the presence of 1 mM Ca2+ (Bonsor et al., 2007), recruitment of TolB in vitro is incomplete resulting in the presence of residual TolB–Pal (peak 2) when the TolB–Pal heterodimer is mixed with TE91-61::DNase stoichiometrically.


The crystal structure of the TolB box of colicin A in complex with TolB reveals important differences in the recruitment of the common TolB translocation portal used by group A colicins.

Zhang Y, Li C, Vankemmelbeke MN, Bardelang P, Paoli M, Penfold CN, James R - Mol. Microbiol. (2009)

ColA binds to TolB but does not competitively recruit TolB from a TolB–Pal complex. A. Analytical gel filtration showing the individual protein peaks attributed to TolB, TE91-61::DNase and TE91-61::DNase–TolB complex. B. Analytical gel filtration showing the individual protein peaks attributed to TolB, TA1–107 and TA1–107–TolB complex. C. Analytical gel filtration showing the protein peaks attributed to Pal, TE91-61::DNase, the TolB–Pal and TE91-61::DNase–TolB complexes, and the peaks produced from a mixture of TolB–Pal incubated stoichiometrically with TE91-61::DNase that shows the displacement of Pal (peak 3) as TolB is competitively recruited by TE91-61::DNase (peak 1). Residual TolB–Pal (peak 2) remains due to incomplete recruitment of TolB in vitro. D. In contrast when TolB and Pal were mixed together, incubated stoichiometrically with TA1–107, and run on gel filtration the absence of a protein peak with the same retention time as TA1–107–TolB demonstrates no competitive recruitment of TolB by TA1–107. Protein peaks attributed to Pal, TA1–107, TolB–Pal and TA1–107–TolB are shown.
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fig05: ColA binds to TolB but does not competitively recruit TolB from a TolB–Pal complex. A. Analytical gel filtration showing the individual protein peaks attributed to TolB, TE91-61::DNase and TE91-61::DNase–TolB complex. B. Analytical gel filtration showing the individual protein peaks attributed to TolB, TA1–107 and TA1–107–TolB complex. C. Analytical gel filtration showing the protein peaks attributed to Pal, TE91-61::DNase, the TolB–Pal and TE91-61::DNase–TolB complexes, and the peaks produced from a mixture of TolB–Pal incubated stoichiometrically with TE91-61::DNase that shows the displacement of Pal (peak 3) as TolB is competitively recruited by TE91-61::DNase (peak 1). Residual TolB–Pal (peak 2) remains due to incomplete recruitment of TolB in vitro. D. In contrast when TolB and Pal were mixed together, incubated stoichiometrically with TA1–107, and run on gel filtration the absence of a protein peak with the same retention time as TA1–107–TolB demonstrates no competitive recruitment of TolB by TA1–107. Protein peaks attributed to Pal, TA1–107, TolB–Pal and TA1–107–TolB are shown.
Mentions: It has recently been shown that the binding surfaces of ColE9 and Pal with TolB are identical and that, in the presence of Ca2+, the TolB box of ColE9 competitively recruits TolB from Pal, presumably as a means of destabilizing the outer membrane on route to cell killing (Loftus et al., 2006). The Kd value of 1.6 µM for binding of TA1–107 to TolB in the presence of Ca2+ is much higher than the value of 90 nM reported for the binding of Pal to TolB in the presence of Ca2+ (Loftus et al., 2006), and thus makes it unlikely that the TolB box of ColA will competitively recruit TolB from the TolB–Pal complex. We confirmed this prediction by using analytical gel filtration chromatography. An interaction was observed between TolB and TE91-61::DNase (Fig. 5A), which is a chimeric polypeptide consisting of the first 61 residues of ColE9 fused to the DNase domain (Macdonald et al., 2004), or TA1–107 (Fig. 5B). However, when the pre-formed TolB–Pal complex was incubated, in the presence of Ca2+, with an equivalent concentration of TE91-61::DNase (Fig. 5C), or TA1–107 (Fig. 5D), only the ColE9 NDR was able to competitively recruit TolB as shown by a retention peak (peak 1) that overlapped the retention peak produced by TE91-61::DNase–TolB, and a retention peak (peak 3) of free Pal (Fig. 5C). As TE91-61::DNase–TolB and TolB–Pal have affinities of binding of 84 nM and 90 nM respectively, in the presence of 1 mM Ca2+ (Bonsor et al., 2007), recruitment of TolB in vitro is incomplete resulting in the presence of residual TolB–Pal (peak 2) when the TolB–Pal heterodimer is mixed with TE91-61::DNase stoichiometrically.

Bottom Line: Comparison of this structure with that of the colicin E9 (ColE9) TolB box-TolB complex, together with site-directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB.Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA.This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection, Immunity and Inflammation, School of Molecular Medical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Interaction of the TolB box of Group A colicins with the TolB protein in the periplasm of Escherichia coli cells promotes transport of the cytotoxic domain of the colicin across the cell envelope. The crystal structure of a complex between a 107-residue peptide (TA(1-107)) of the translocation domain of colicin A (ColA) and TolB identified the TolB box as a 12-residue peptide that folded into a distorted hairpin within a central canyon of the beta-propeller domain of TolB. Comparison of this structure with that of the colicin E9 (ColE9) TolB box-TolB complex, together with site-directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB. Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA. This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.

Show MeSH
Related in: MedlinePlus