Abstract
The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide’s stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide.
Highlights
The inhibitor cystine knot (ICK) is a protein scaffold defined as an antiparallel sheet stabilized by a cystine knot [1,2]
In order to determine whether the degradation observed at high temperature is reversible, we incubated a sample of Hv1a at 95 °C for 24 h, stored the sample at 20 °C for 3 days prior to HPLC analysis
We demonstrated that the insecticidal spider-venom peptide Hv1a is remarkably resistant to chemical degradation; the peptide is stable over the pH range 1–8, at temperatures up to 75 °C, and when dissolved in a range of organic solvents
Summary
The inhibitor cystine knot (ICK) is a protein scaffold defined as an antiparallel sheet stabilized by a cystine knot [1,2]. The cystine knot itself comprises a ring formed by two disulfide bridges (Cys1–Cys and Cys2–Cys5) and the intervening sections of peptide backbone, with a third disulfide bond (Cys3–Cys6) penetrating the ring to create a pseudo-knot (Figure 1B). In the present study, we used the insecticidal spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) as a model ICK peptide and explored the contribution of the cystine knot to its physicochemical stability. Reduction and alkylation of the six cysteine residues that form the cystine knot motif of Hv1a completely abrogated the peptide’s resistance to enzymatic degradation and its resistance to high temperature and acidic pH. This study reveals that the ICK motif is capable of providing spider-venom peptides with a high level of thermal, chemical and biological stability, and it further highlights the ICK motif as an excellent scaffold for protein engineering studies directed towards the development of peptide drugs and bioinsecticides
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