Cell entry mechanism of enzymatic bacterial colicins: porin recruitment and the thermodynamics of receptor binding

Abstract

Binding of enzymatic E colicins to the vitamin B12 receptor, BtuB, is the first stage in a cascade of events that culminate in the translocation of the cytotoxic nuclease into the Escherichia coli cytoplasm and release of its tightly bound immunity protein. A dogma of colicin biology is that the toxin coiled-coil connecting its functional domains must unfold or unfurl to span the periplasm, with recent reports claiming this reaction is initiated by receptor binding. We report isothermal titration calorimetry data of BtuB binding the endonuclease toxin ColE9 and a disulfide form (ColE9S-S) where unfolding of the coiled-coil is prevented and, as a consequence, the toxin is biologically inactive. Contrary to expectation, the thermodynamics of receptor binding, characterized by large negative values for TDeltaS, are identical for the two colicins, arguing against any form of BtuB-induced unfolding. We go on to delineate key features of the "colicin translocon" that assembles at the cell surface after BtuB binding by using a complex of histidine-tagged Im9 bound to ColE9S-S. First, we show that the porin OmpF is recruited directly to the BtuB.colicin complex to form the translocon. Second, recruitment is through the natively unfolded region of the colicin translocation domain, with this domain likely having two contact points for OmpF. Finally, the immunity protein is not released during its assembly. Our study demonstrates that although colicin unfolding is undoubtedly a prerequisite for E. coli cell death, it must occur after assembly of the translocon.

Fig. 1.

Structure of ColE3/Im3 modeled on that of the BtuB·ColE3 R-domain complex (11, 12). The figure highlights the positions of the R and T domains of the enzymatic E colicin, the C-terminal E3 rRNase bound to Im3, and the locations of the T domain deletions that were engineered into an R-domain disulfide bond variant of ColE9 (ColE9^S-S). (Inset) Structure of the ColE9 DNase/Im9 complex (13). The asterisk shows where on the full-length colicin the domain would be attached in ColE9/Im9. ColE3 and ColE9 are 92% identical in sequence up to this point.

Fig. 2.

Representative ITC binding curves for the formation of the BtuB·ColE9/Im9 complex. (a) Titration of a 16 μM ColE9/Im9 complex into 1.2 μM BtuB in 25 mM Tris·HCl, pH 7.5/150 mM NaCl/1% β-OG in the presence of 5 mM EGTA. (b) Equivalent titration of 16 μM ColE9/Im9 complex into 1.2 μM BtuB in 25 mM Tris·HCl, pH 7.5/150 mM NaCl/1% β-OG in the presence of 5 mM CaCl₂. Data were fitted to a single binding-site model. Derived thermodynamic parameters for these complexes are listed in Table 1.

Fig. 3.

OmpF is recruited to the BtuB·ColE9^S-S/Im9 complex by the disordered colicin T domain. Outer membrane protein complexes were extracted from E. coli B^E3000 membranes and purified in the presence of full length ColE9^S-S/Im9 (a), Δ1–316 ColE9^S-S/Im9 (b) or Δ1–83 ColE9^S-S/Im9 (c) and loaded onto 1-ml Ni-charged HisTrap columns equilibrated in 20 mM Tris·HCl, pH 8.0/25 mM NaCl/5 mM imidazole/1% (wt/vol) β-OG. The chromatograms show protein complexes eluted from the column with a 5–300 mM imidazole gradient, while monitoring the absorbance of the eluent at 280 nm, and have been corrected for buffer contributions. In a and c, single peaks were observed, whereas in b, two peaks eluted from the column, the latter most likely due to a weakening of the interaction between ColE9 and BtuB in this deletion construct. The 10% SDS-polyacrylamide gels adjacent to each chromatogram include the peak fraction(s) from elution, the outer membrane extract loaded onto each column, and controls of full-length or truncated colicins, BtuB and OmpF. In all cases, BtuB and ColE9 were recovered but OmpF was lost when the colicin lacked the disordered T domain. With the low percentage SDS-polyacrylamide gels used to resolve extracted proteins, Im9 was not separated from the dye front and is not observed. Bands in the eluted fractions were excised, digested with trypsin, and the proteins identified unambiguously by MALDI-TOF mass spectrometry (data not shown).

Fig. 4.

Colicins deficient in OmpF recruitment are less effective at killing E. coli K-12 cells. ColE9^S-S/Im9 and the T-domain deletions Δ1–7, Δ1–30, and Δ1–60 ColE9^S-S/Im9 were added to separate flasks of E. coli JM83 cells at the point of inoculation (0 h). All cultures behaved identically until the addition of DTT (5 mM) when the cultures had reached an optical density of ≈0.5 (as indicated by the arrow), reducing the R-domain disulfide bond and, hence, initiating cell killing. Δ1–60 was inactive in this assay, whereas full-length ColE9^S-S/Im9 showed wild-type colicin cytotoxic activity. Δ1–7 and Δ1–30 ColE9^S-S/Im9 constructs had identical cell-killing profiles that were significantly reduced relative to full-length Col.