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

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.

Fig. 1

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.

Fig. 2

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 TA_1–107 on the TA_1–107–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⁺.

Fig. 3

Structure of the TA_1–107–TolB complex. A. Electron density map of residues 9–20 of TA_1–107 contoured at 0.95 σ. Residues 9–20 were the only ColA residues with any electron density in the TA_1–107–TolB cocrystal structure. Note, contouring was shown at 0.95 σ rather than 1 σ to provide a sharper representation of the electron density map given the resolution of the data. B. Structure of the TA_1–107–TolB complex at 2.6 Å resolution showing the colicin binding site of the β-propeller domain. Also visible are one Ca²⁺ ion (blue) in the central channel of the β-propeller domain and one Na⁺ ion (violet) between the β-propeller and N-terminal α/β-domains. C. Intermolecular hydrogen bonding networks in the core region of the proximal half of the TA_1–107–TolB peptide complex. ColA residues are shown in blue and the TolB residues are shown in green. The intermolecular hydrogen bonds are shown as black lines, three of which are mediated by water molecules.

Fig. 4

Comparison of TA_1–107–TolB with TE9_pep32–47–TolB (PDB entry 2IVZ). A. Ball and stick comparison of the stereochemistry of the core region of the TolB box of ColA (blue) with ColE9 (green). Intramolecular hydrogen bonds are shown in magenta dashed lines for ColE9 and black dashed lines for ColA. The hydrogen bond between S37 and S40 of TE9_pep32–47–TolB (shown by the arrow) has no equivalence in TA_1–107–TolB due to the small perturbation in the position of the T13 residue. B. Ball and stick representation of residues 9–20 of TA_1–107 bound to the central canyon of the TolB β-propeller (left) in comparison with the TE9_pep32–47–TolB interaction (right). The cyclizing hydrogen bond between G32 and N44 in ColE9 is shown as a green dashed line.

Fig. 5

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, TE9_1-61::DNase and TE9_1-61::DNase–TolB complex. B. Analytical gel filtration showing the individual protein peaks attributed to TolB, TA_1–107 and TA_1–107–TolB complex. C. Analytical gel filtration showing the protein peaks attributed to Pal, TE9_1-61::DNase, the TolB–Pal and TE9_1-61::DNase–TolB complexes, and the peaks produced from a mixture of TolB–Pal incubated stoichiometrically with TE9_1-61::DNase that shows the displacement of Pal (peak 3) as TolB is competitively recruited by TE9_1-61::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 TA_1–107, and run on gel filtration the absence of a protein peak with the same retention time as TA_1–107–TolB demonstrates no competitive recruitment of TolB by TA_1–107. Protein peaks attributed to Pal, TA_1–107, TolB–Pal and TA_1–107–TolB are shown.

Fig. 6

Competitive recruitment of TolB by ColA containing the TolB box of ColE9 (YZ78). A. Gel filtration of a mixture of TolB and Pal incubated stoichiometrically with YZ78 in the presence of 1 mM Ca²⁺. Peak 1 of the YZ78–TolB–Pal mixture superimposes with the YZ78–TolB control. B. Fractions collected across peaks 1–4 of the YZ78–TolB–Pal retention profile in A were analysed by SDS-PAGE. Peak 1 contained TolB and YZ78 indicating good separation of YZ78–TolB from any TolB–Pal. Peak 2 is a shoulder peak and contained a mixture of YZ78–TolB and TolB–Pal. Peak 3 contained uncomplexed YZ78, and peak 4 contained free Pal that had been displaced from the TolB–Pal interaction. *Indicates some breakdown product of YZ78 in the uncomplexed fractions.

Fig. 7

ColA expressing the TolB box of ColE9 (YZ73) has at least 100-fold less biological activity than ColA. Cell killing of E. coli DH5α in liquid culture following treatment with 0.1–10 nM of colicin A (A) and 10 nM to 1 µM of YZ73 (B). An untreated sample was included as a negative control for cell killing of both ColA and YZ73. Comparison of both panels suggests that 0.1 nM and 10 nM ColA had similar killing properties to 10 nM and 1 µM YZ73 respectively, while 1 nM of ColA had a killing activity similar to 500 nM of YZ73 indicating a reduced activity of YZ73 of, at least, 100-fold when compared with ColA.