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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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Alanine scanning mutagenesis of residues of the TolB box of ColA. A. Individual alanine mutations were engineered into ColA from Lys9 to Pro25 and their effect on the activity of ColA was determined using the spot test assay using doubling dilutions of purified proteins from 25 nM to 0.1 nM. B. The effect of each TolB box mutant of TA1–107 on the TA1–107–TolB interaction was determined by surface plasmon resonance and expressed in response units (RU) in the presence (grey bars) and absence (black bars) of 1 mM Ca2+.
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fig02: Alanine scanning mutagenesis of residues of the TolB box of ColA. A. Individual alanine mutations were engineered into ColA from Lys9 to Pro25 and their effect on the activity of ColA was determined using the spot test assay using doubling dilutions of purified proteins from 25 nM to 0.1 nM. B. The effect of each TolB box mutant of TA1–107 on the TA1–107–TolB interaction was determined by surface plasmon resonance and expressed in response units (RU) in the presence (grey bars) and absence (black bars) of 1 mM Ca2+.

Mentions: Alanine scanning mutagenesis was conducted across the region from residue K9 to P25 to determine the extent of the TolB box of ColA and the contribution of individual residues to the binding of TolB. The biological activity of the alanine mutants in comparison with ColA (Fig. 2A) indicates that mutations in residues D11, T13, W15 or E18 abolished the biological activity of the mutant colicin. In contrast, alanine mutations of residues G12, G14, R19, G20 or G22 had little effect on biological activity while alanine mutations of residues S17 and S21 had no effect, and thus also presumably contribute little to the affinity of binding to TolB. We are, however, aware that the N44A mutation in the TolB box of ColE9 that abolished biological activity did not affect binding of the mutant colicin to TolB (Hands et al., 2005). Consequently, we determined the interaction of the ColA TolB box containing peptide (TA1–107) and all the TA1–107 alanine mutants, from K9 to P25 with TolB by SPR, in the presence or absence of Ca2+ (Fig. 2B), as Ca2+ has been shown previously to dramatically increase the binding affinity of the ColE9 TolB box for TolB (Loftus et al., 2006). Corrected sensorgrams and residual plots for the interaction of TolB with ColA and ColA E18A are shown in Fig. S1. In the presence of Ca2+ the affinity of binding (Kd) of TA1–107 to TolB was 1.6 µM, compared with a value of 24.3 µM in the absence of Ca2+, indicating a similar enhancement by Ca2+ of the binding of ColA to TolB. Compared with TA1–107, alanine substitutions of residues K9 and S21 to P25 did not affect binding to TolB, whereas alanine substitutions of all residues between G10 and G20, with the exception of G14, resulted in either complete inhibition or significantly reduced binding to TolB when compared with that of ColA (Fig. 2). Interestingly, the ColA S16A mutant had partial biological activity using the spot test assay but a binding affinity to TolB that was very weak and similar to the inactive mutants, D11A, T13A, W15A and E18A. As the spot test assay only measures the activity of a colicin at the end of a 16 h incubation period, it is possible that this endpoint assay does not always fully differentiate between complexes that have different affinities or stabilities. Indeed, analysis of the biological activity of ColA containing the S16A mutation using a liquid growth assay over an 8 h period showed that the S16A mutation reduced the activity of ColA by, at least, 1000-fold compared with ColA, thus confirming the SPR data (Fig. S2). This datum indicates that the TolB box of ColA is located between residues G10 and G20 with many of the core residues being highly conserved between the tol-dependent enzymatic and pore-forming colicins (Fig. 1).


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)

Alanine scanning mutagenesis of residues of the TolB box of ColA. A. Individual alanine mutations were engineered into ColA from Lys9 to Pro25 and their effect on the activity of ColA was determined using the spot test assay using doubling dilutions of purified proteins from 25 nM to 0.1 nM. B. The effect of each TolB box mutant of TA1–107 on the TA1–107–TolB interaction was determined by surface plasmon resonance and expressed in response units (RU) in the presence (grey bars) and absence (black bars) of 1 mM Ca2+.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2821528&req=5

fig02: Alanine scanning mutagenesis of residues of the TolB box of ColA. A. Individual alanine mutations were engineered into ColA from Lys9 to Pro25 and their effect on the activity of ColA was determined using the spot test assay using doubling dilutions of purified proteins from 25 nM to 0.1 nM. B. The effect of each TolB box mutant of TA1–107 on the TA1–107–TolB interaction was determined by surface plasmon resonance and expressed in response units (RU) in the presence (grey bars) and absence (black bars) of 1 mM Ca2+.
Mentions: Alanine scanning mutagenesis was conducted across the region from residue K9 to P25 to determine the extent of the TolB box of ColA and the contribution of individual residues to the binding of TolB. The biological activity of the alanine mutants in comparison with ColA (Fig. 2A) indicates that mutations in residues D11, T13, W15 or E18 abolished the biological activity of the mutant colicin. In contrast, alanine mutations of residues G12, G14, R19, G20 or G22 had little effect on biological activity while alanine mutations of residues S17 and S21 had no effect, and thus also presumably contribute little to the affinity of binding to TolB. We are, however, aware that the N44A mutation in the TolB box of ColE9 that abolished biological activity did not affect binding of the mutant colicin to TolB (Hands et al., 2005). Consequently, we determined the interaction of the ColA TolB box containing peptide (TA1–107) and all the TA1–107 alanine mutants, from K9 to P25 with TolB by SPR, in the presence or absence of Ca2+ (Fig. 2B), as Ca2+ has been shown previously to dramatically increase the binding affinity of the ColE9 TolB box for TolB (Loftus et al., 2006). Corrected sensorgrams and residual plots for the interaction of TolB with ColA and ColA E18A are shown in Fig. S1. In the presence of Ca2+ the affinity of binding (Kd) of TA1–107 to TolB was 1.6 µM, compared with a value of 24.3 µM in the absence of Ca2+, indicating a similar enhancement by Ca2+ of the binding of ColA to TolB. Compared with TA1–107, alanine substitutions of residues K9 and S21 to P25 did not affect binding to TolB, whereas alanine substitutions of all residues between G10 and G20, with the exception of G14, resulted in either complete inhibition or significantly reduced binding to TolB when compared with that of ColA (Fig. 2). Interestingly, the ColA S16A mutant had partial biological activity using the spot test assay but a binding affinity to TolB that was very weak and similar to the inactive mutants, D11A, T13A, W15A and E18A. As the spot test assay only measures the activity of a colicin at the end of a 16 h incubation period, it is possible that this endpoint assay does not always fully differentiate between complexes that have different affinities or stabilities. Indeed, analysis of the biological activity of ColA containing the S16A mutation using a liquid growth assay over an 8 h period showed that the S16A mutation reduced the activity of ColA by, at least, 1000-fold compared with ColA, thus confirming the SPR data (Fig. S2). This datum indicates that the TolB box of ColA is located between residues G10 and G20 with many of the core residues being highly conserved between the tol-dependent enzymatic and pore-forming colicins (Fig. 1).

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