Abstract
Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P’s structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with 1H–15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second.
Highlights
L ow-pH-induced conformational change is a feature of many infectious processes
The first transition moved toward a lower urea concentration, while the second transition moved to higher concentrations such that, eventually, it did not unfold within the urea concentration range used
The shift in the second transition to a high urea concentration appears complete at pH 4.5, but because the full transition is not observed within the urea concentration range that is used, it is unclear whether this continues to change at even lower pH values
Summary
L ow-pH-induced conformational change is a feature of many infectious processes. Some involve pore-forming toxins such as anthrax[1] and diphtheria,[2−4] but influenza infection is pH-dependent due to the need for the hemagglutinin protein to undergo low-pH-induced conformational change.[5,6] Both hemagglutinin and protein toxins must remain as water-soluble proteins until required to enter the membrane, and the low pH of the late endosome supplies the specific trigger for activation. Colicins are bacterial protein toxins that kill Gram-negative bacteria such as E. coli by translocating across the outer membrane to deliver a toxic C-terminal domain, with pore forming or nuclease activity, into the cytoplasmic membrane.[12,13] In pore-forming colicins, these domains enter the energized inner membrane and depolarize it by ion release.[14] The water-soluble pore-forming domains of colicins are conserved 10-helix bundles, similar to Bcl2-family apoptosis regulators,[15] which contain a buried hydrophobic helical hairpin that is important in membrane insertion and channel formation These domains are stable folded proteins so their tertiary structure needs to be destabilized to enable the unfolding that must precede membrane insertion.[16]. The stabilizing colicin A aspartates do not all fit into any one of these categories, so how they show similar pH-dependent effects was unclear
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