Antibacterial toxin colicin N and phage protein G3p compete with TolB for a binding site on TolA

Abstract

Most colicins kill Escherichia coli cells by membrane pore formation or nuclease activity and, superficially, the mechanisms are similar: receptor binding, translocon recruitment, periplasmic receptor binding and membrane insertion. However, in detail, they employ a wide variety of molecular interactions that reveal a high degree of evolutionary diversification. Group A colicins bind to members of the TolQRAB complex in the periplasm and heterotrimeric complexes of colicin-TolA-TolB have been observed for both ColA and ColE9. ColN, the smallest and simplest pore-forming colicin, binds only to TolA and we show here that it uses the binding site normally used by TolB, effectively preventing formation of the larger complex used by other colicins. ColN binding to TolA was by β-strand addition with a KD of 1 µM compared with 40 µM for the TolA-TolB interaction. The β-strand addition and ColN activity could be abolished by single proline point mutations in TolA, which each removed one backbone hydrogen bond. By also blocking TolA-TolB binding these point mutations conferred a complete tol phenotype which destabilized the outer membrane, prevented both ColA and ColE9 activity, and abolished phage protein binding to TolA. These are the only point mutations known to have such pleiotropic effects and showed that the TolA-TolB β-strand addition is essential for Tol function. The formation of this simple binary ColN-TolA complex provided yet more evidence of a distinct translocation route for ColN and may help to explain the unique toxicity of its N-terminal domain.

Fig. 1.

(a) Secondary structure analysis of ColN-T_1–90. Structural predictions. Primary sequence of the ColN-T TABS, residues 40–74; there was no prediction of structure elsewhere. Previously reported binding to TolA_III (Anderluh et al., 2004; Gokce et al., 2000; Raggett et al., 1998) is shown as WT binding (<10 µM) (red squares), intermediate binding (green diamonds) and no binding (blue circles). The self-recognition region determined by NMR by Hecht et al. (2008) is highlighted in grey. Schematic representation of the secondary structure predictions by the i-tasser three-dimensional model (Zhang, 2008) (top line), Jpred algorithm (Cole et al., 2008) (middle line) and talos prediction (Cornilescu et al., 1999) of the β-structure using the backbone chemical shifts of the T domain bound to TolA_III (bottom line). (b) i-tasser ribbon model of ColN-T_1–90; coloured regions as in (a). (c) Binding of ColA-T (red) and G3p-N1 (green) to TolA_III. Drawn in PyMOL (http://www.pymol.org/) using PDB ID: 3QDR and PDB ID: 1Tol, respectively. The ColA and G3p β-strand binding regions are shown in brown and magenta, respectively. Site of G3p-N1 V44P mutation shown in yellow. (d) Primary structure of TolA_III S333–P421. Secondary structure elements: α-helices (red blocks) and β-strands (blue arrows). Contacts with G3p-N1, inter-atomic distances <4.5 Å (yellow dots) (PDB ID: 1Tol; Lubkowski et al., 1999). Residues which showed the strongest NMR chemical shift variations (>0.55 p.p.m.) upon binding ColN-T_1–90 (Hecht et al., 2009) are shown as red dots. The ColA-binding box (Li et al., 2012) is shown. The residues mutated in this study are shaded pink. The ColA (β2) and G3p (β3) β-strand binding regions are shown in brown and magenta, respectively.

Fig. 2.

The effect of mutations in TolA_III on protein structure and colicin sensitivity. (a) A selection of spot test assays showing the colicin sensitivity of TolA mutants compared with WT pSKL10, an empty pUC19 vector control and ΔTolA E. coli cells. Concentrations of 10, 5, 1, 0.5, 0.1 and 0 µM ColN (left) or ColA (right) (in 50 mM sodium phosphate, pH 7.5, 300 mM NaCl) were spotted (2 µl) onto a lawn of E. coli JC207 ΔTolA cells complemented with pSKL10 WT TolA or mutant plasmids. (b) A selection of growth curves at 30 °C of JC207 ΔTolA cells complemented with pSKL10 WT or mutant TolA plasmid. Data are shown as WT TolA_III (black), pUC19 (negative control, white), JC207 cells only (magenta), Y340A (blue), I344D (orange), A415R (cyan) and F419A (green). A 10 nM final concentration of ColN was added after 273 min (arrow); growth continued for 153 min after which time the OD₆₀₀ was used to calculate the percentage of killing using TolA_III WT and JC207 cells only to set 100 and 0 % killing, respectively (see Table 1). (c) A representative selection of far-UV CD spectra of TolA_III variants, measured in a 0.2 mm path-length cuvette at 25 °C. The protein concentration was typically 40 µM in 20 mM sodium phosphate, pH 7. Data are shown as WT TolA_III (black), Y340A (blue), I344D (orange), A415R (cyan) and F419A (green).

Fig. 3.

Mutations in TolA_III. (a) Cartoon representation of the TolA_III domain showing sites of proline insertion in β3 (D418P, red; K420P, green). Orange region shows the ColA-binding site from Li et al. (2012). (b) Backbone representation of β1–3 showing mutation sites and internal hydrogen bonding. Backbone amide groups removed by proline mutations are highlighted. (c) Far-UV CD spectra of TolA_III variants. CD spectra were measured in a 0.2 mm path-length cuvette at 25 °C. The protein concentration was typically 43 µM in 20 mM sodium phosphate, pH 7. Data are shown as WT TolA_III (solid line), D418P (dotted line), D418V (dot-dashed line) and K420P (dashed line). Inset: normalized far-UV CD (222 nm) thermal denaturation profile (1 °C min⁻¹) of TolA_III (○), D418P (•), D418V (▴) and K420P (□) at ~0.14 mg ml⁻¹ in 20 mM sodium phosphate, pH 7.0. (d) Table showing derived T_m from far-UV CD (222 nm) thermal denaturation and SDS sensitivity of TolA_III variants (see Fig. S3). (e) Spot test assay showing sensitivity of mutants to ColN (method as in Fig. 2).

Fig. 4.

Biacore binding data of ColN-T, ColA-T, G3p-N1 and TolB to WT TolA_III, D418P and K420P. Around 500 RU of either (a) TolA_III276–421WT, (b) D418P or (c) K420P protein was immobilized on a CM5 sensor chip using amine coupling. ColN-T (solid line), ColA-T (dotted line) or G3p-N1 (dashed line) (5 µM) were injected over each surface at 30 µl min⁻¹ for 120 s followed by regeneration with 10 mM glycine, pH 1.8. (d) TolB (10 µM in HEPES-EP buffer) was injected over the immobilized WT and TolA_III mutant surfaces described above at 10 µl min⁻¹ for 180 s followed by regeneration with 10 mM glycine, pH 1.8. Only the WT surface bound TolB. (e) WT and mutants of TolB (5 µM) were injected over immobilized TolA_III. WT TolB (solid line), DGSY-AGAA (dash-dotted line), V101P (dotted line) and VVV-AAA (dashed line). In each case, a control surface which had no immobilized protein was subtracted from the data. Data represent the means of duplicate injections.