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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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Alignment of residues of the TolB box region of pore-forming and enzymatic group A colicins. Residues of the extended TolB box of ColE9 and residues of the TolB box sequence that are conserved in the other colicin sequences are shown in bold. The residue numbers are indicated at the start and end of each sequence. A padding space has been introduced in the ColY sequence to optimize the alignment. Colicins A, U, Y and E2-E9 are produced by E. coli, Col28b is produced by Serratia marcescens, Klebicin D is from Erwinia tasmaniensis and Alveicin A is from Hafnia alvei.
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fig01: Alignment of residues of the TolB box region of pore-forming and enzymatic group A colicins. Residues of the extended TolB box of ColE9 and residues of the TolB box sequence that are conserved in the other colicin sequences are shown in bold. The residue numbers are indicated at the start and end of each sequence. A padding space has been introduced in the ColY sequence to optimize the alignment. Colicins A, U, Y and E2-E9 are produced by E. coli, Col28b is produced by Serratia marcescens, Klebicin D is from Erwinia tasmaniensis and Alveicin A is from Hafnia alvei.

Mentions: The TolB box in ColE9 consists of 15 contiguous residues with the sequence 32-GASDGSGWSSENNPW-46 (Hands et al., 2005) (Fig. 1). Using deletion analysis, the TolB box of ColA was predicted to include residues 7–20 (Bouveret et al., 1998; Journet et al., 2001). Sequence alignment of the tol-dependent colicins showed that the DG(S/T)GWSSE residues are highly conserved in all of the enzymatic and pore-forming colicins (shown in bold in Fig. 1). Previous mutagenesis studies have shown that the G38 residue of ColE9 is not essential for activity (Garinot-Schneider et al., 1997), and that the S37 residue of ColE9 can be substituted by threonine without significant loss of function (Hands et al., 2005). We therefore assume that substitution of G14 of ColA to N14 or S14 of Col28b and Klebicin D respectively, and G36 of ColE9 to N34 of Alveicin A would similarly not affect biological activity. It is intriguing that ColA and the other pore-forming colicins are missing the last four residues of the extended TolB box of ColE9 (43-NNPW-46) which are replaced by RGSG in ColA. We have previously demonstrated that the N44A and W46A mutations resulted in loss of biological activity of ColE9, with the latter mutation abolishing the interaction with TolB (Hands et al., 2005).


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)

Alignment of residues of the TolB box region of pore-forming and enzymatic group A colicins. Residues of the extended TolB box of ColE9 and residues of the TolB box sequence that are conserved in the other colicin sequences are shown in bold. The residue numbers are indicated at the start and end of each sequence. A padding space has been introduced in the ColY sequence to optimize the alignment. Colicins A, U, Y and E2-E9 are produced by E. coli, Col28b is produced by Serratia marcescens, Klebicin D is from Erwinia tasmaniensis and Alveicin A is from Hafnia alvei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Alignment of residues of the TolB box region of pore-forming and enzymatic group A colicins. Residues of the extended TolB box of ColE9 and residues of the TolB box sequence that are conserved in the other colicin sequences are shown in bold. The residue numbers are indicated at the start and end of each sequence. A padding space has been introduced in the ColY sequence to optimize the alignment. Colicins A, U, Y and E2-E9 are produced by E. coli, Col28b is produced by Serratia marcescens, Klebicin D is from Erwinia tasmaniensis and Alveicin A is from Hafnia alvei.
Mentions: The TolB box in ColE9 consists of 15 contiguous residues with the sequence 32-GASDGSGWSSENNPW-46 (Hands et al., 2005) (Fig. 1). Using deletion analysis, the TolB box of ColA was predicted to include residues 7–20 (Bouveret et al., 1998; Journet et al., 2001). Sequence alignment of the tol-dependent colicins showed that the DG(S/T)GWSSE residues are highly conserved in all of the enzymatic and pore-forming colicins (shown in bold in Fig. 1). Previous mutagenesis studies have shown that the G38 residue of ColE9 is not essential for activity (Garinot-Schneider et al., 1997), and that the S37 residue of ColE9 can be substituted by threonine without significant loss of function (Hands et al., 2005). We therefore assume that substitution of G14 of ColA to N14 or S14 of Col28b and Klebicin D respectively, and G36 of ColE9 to N34 of Alveicin A would similarly not affect biological activity. It is intriguing that ColA and the other pore-forming colicins are missing the last four residues of the extended TolB box of ColE9 (43-NNPW-46) which are replaced by RGSG in ColA. We have previously demonstrated that the N44A and W46A mutations resulted in loss of biological activity of ColE9, with the latter mutation abolishing the interaction with TolB (Hands et al., 2005).

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