Helix N‑Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A

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.