Anthony H. Keeble

The cytotoxic domain of colicin E9 is a channel-forming endonuclease.

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.

Distinct conformational stability and functional activity of four highly homologous endonuclease colicins

The family of conserved colicin DNases E2, E7, E8, and E9 are microbial toxins that kill bacteria through random degradation of the chromosomal DNA. In the present work, we compare side by side the conformational stabilities of these four highly homologous colicin DNases. Our results indicate that the apo‐forms of these colicins are at room temperature and neutral pH in a dynamic conformational equilibrium between at least two quite distinct conformers. We show that the thermal stabilities of the apo‐proteins differ by up to 20°C. The observed differences correlate with the observed conformational behavior, that is, the tendency of the protein to form either an open, less stable or closed, more stable conformation in solution, as deduced by both tryptophan accessibility studies and electrospray ionization mass spectrometry. Given these surprising structural differences, we next probed the catalytic activity of the four DNases and also observed a significant variation in relative activities. However, no unequivocal link between the activity of the protein and its thermal and structural stability could easily be made. The observed differences in conformational and functional properties of the four colicin DNases are surprising given that they are a closely related (≥65% identity) family of enzymes containing a highly conserved (ββα‐Me) active site motif. The different behavior of the apo‐enzymes must therefore most likely depend on more subtle changes in amino acid sequences, most likely in the exosite region (residues 72–98) that is required for specific high‐affinity binding of the cognate immunity protein.

Metal induced selectivity in phosphate ion binding in E9 DNase

Mass spectrometric and calorimetric data reveal that phosphate ion binding to the active site of colicin E9 DNase is delicately regulated by concomitant binding of specific transition metal ions.