Directional Porin Binding of Intrinsically Disordered Protein Sequences Promotes Colicin Epitope Display in the Bacterial Periplasm

Abstract

Protein bacteriocins are potent narrow spectrum antibiotics that exploit outer membrane porins to kill bacteria by poorly understood mechanisms. Here, we determine how colicins, bacteriocins specific for Escherichia coli, engage the trimeric porin OmpF to initiate toxin entry. The N-terminal ∼80 residues of the nuclease colicin ColE9 are intrinsically unstructured and house two OmpF binding sites (OBS1 and OBS2) that reside within the pores of OmpF and which flank an epitope that binds periplasmic TolB. Using a combination of molecular dynamics simulations, chemical trimerization, isothermal titration calorimetry, fluorescence microscopy, and single channel recording planar lipid bilayer measurements, we show that this arrangement is achieved by OBS2 binding from the extracellular face of OmpF, while the interaction of OBS1 occurs from the periplasmic face of OmpF. Our study shows how the narrow pores of oligomeric porins are exploited by colicin disordered regions for direction-specific binding, which ensures the constrained presentation of an activating signal within the bacterial periplasm.

Figure 1

ColE9 IUTD with the TBE flanked by OBS1 and OBS2. Binding of two OBSs to OmpF presents the TBE in the periplasm. OBSs are represented with arrowheads at N-termini to highlight the direction of binding.

Figure 2

TMEA mediated trimerization of T25C ColE9^1–32-DNase (OBS1) and S71C ColE9^53–83-DNase (OBS2) yields high affinity tridentate OmpF ligands. (A) Cartoon representation of OBS DNase fusion proteins in their monomeric and tridentate forms. Sequences of OBS1 and OBS2 represented by yellow and brown arrows, respectively, are shown. (B) Titration of 120 μM OBS1 (○) and 40 μM (OBS1)₃ (●) into 4 μM OmpF trimer, in 20 mM potassium phosphate buffer, pH 6.5, 1% (w/v) β-OG. When fitted to a single set of identical sites binding model, the tridentate complex gives a K_d of 29 ± 2 nM, N = 3.0 ± 0.3, and ΔH = −26.7 ± 2.1 kcal·mol^–1, compared to the previously published values of K_d = 2.5 μM, N = 2.5, and ΔH = −26.7 kcal·mol^–1 for the binding of OBS1 to OmpF under identical conditions. (C) Titration of 375 μM OBS2 (□) and 125 μM (OBS2)₃ (■) into 11 μM OmpF trimer, in 20 mM potassium phosphate buffer, pH 6.5, 1% (w/v) β-OG. Data were fitted to a single set of identical sites binding model to give K_d = 480 ± 5 nM, N = 3.0 ± 0.1, and ΔH = −5.64 ± 0.01 kcal·mol^–1, compared to previously published values of K_d = 134 μM, N = 2.6, and ΔH = −27.6 kcal·mol^–1 for the binding of OBS2 under identical conditions.

Figure 3

Confocal microscopy data showing tridentate OBS1 binds OmpF from the periplasm while tridentate OBS2 binds from the extracellular medium. (OBS1)₃^TMR only bound to OmpF when E. coli B^E3000 cells were permeabilized (compare columns 1 and 2), whereas (OBS2)₃^TMR could bind intact cells (column 4), suggesting the two OBSs associate with OmpF in opposite directions. No staining of BZB1107 ompF^– cells was observed in the presence of either (OBS1)₃^TMR or (OBS2)₃^TMR, even when cells where permeabilized. Ligands were added to mid-log cultures of B^E3000 and BZB1107 cells. Following extensive washing, cells were mounted between an agar pad and a coverslip for imaging. Imaging was carried out on at least three regions of interest (21 μm × 21 μm) per condition, with n = 30 cells in each experiment. Quantification of the fluorescence intensities is shown in Figure S4.

Figure 4

Voltage-gated OmpF channels are occluded by (OBS1)₃ added from the periplasmic face of OmpF while (OBS2)₃ occludes from the extracellular face. Electrical recordings from single OmpF trimers incorporated into a DPhPC planar lipid bilayer with 50 nM (OBS1)₃ or (OBS2)₃ added to the cis compartment, corresponding to the extracellular side of the membrane (A and B, respectively), were measured with a holding potential of −100 mV. Equivalent measurements adding 50 nM (OBS1)₃ or (OBS2)₃ to the trans chamber, corresponding to the periplasmic side of the membrane (C and D, respectively), were made at a holding potential of +100 mV. For each experiment, channel recordings are shown over 3 min time courses, with the initial 4 s shown on an expanded scale for the addition of (OBS1)₃ from the periplasmic side of the membrane, where the occlusion of OmpF channels was rapid. In each panel, conductance of the open channel is marked with a gray line, while the closed channel with zero conductance is marked by a red line.

Figure 5

Negative charge within the N-terminus destabilizes OBS1 interaction from the extracellular face of OmpF. (A) Cartoon representation of (OBS1)₃^TMR, (Δ2–5 OBS1)₃^TMR, and (D5A OBS1)₃^TMR added to OmpF from the extracellular face. (B) Confocal fluorescence microscopy of (OBS1)₃^TMR, (Δ2–5 OBS1)₃^TMR, and (D5A OBS1)₃^TMR added to E. coli B^E3000 in the absence of permeabilization of the outer membrane. Quantification of fluorescence intensities is shown in Figure S4. (C) Electrical recording of single OmpF trimers incorporated into DPhPC planar lipid bilayers with (OBS1)₃, (Δ2–5 OBS1)₃, and (D5A OBS1)₃ added to the cis (extracellular) chamber at a holding potential of −100 mV. Data are shown over 125 s time courses with the initial 5 s shown on an expanded scale. Open channel conductance is marked with a gray line, while zero conductance is marked by the red line. (D) MD simulations for the OmpF·OBS1, OmpF·Δ2–5 OBS1, and OmpF·D5A OBS1 complexes with the N-terminus of the peptide modeled facing the periplasm, showing snapshots of the simulation over the 100 ns time course.