Structural Conformation and Activity of Spider-Derived Inhibitory Cystine Knot Peptide Pn3a Are Modulated by pH

Abstract

Numerous spider venom-derived gating modifier toxins exhibit conformational heterogeneity during purification by reversed-phase high-performance liquid chromatography (RP-HPLC). This conformational exchange is especially peculiar for peptides containing an inhibitor cystine knot motif, which confers excellent structural stability under conditions that are not conducive to disulfide shuffling. This phenomenon is often attributed to proline cis/trans isomerization but has also been observed in peptides that do not contain a proline residue. Pn3a is one such peptide forming two chromatographically distinguishable peaks that readily interconvert following the purification of either conformer. The nature of this exchange was previously uncharacterized due to the fast rate of conversion in solution, making isolation of the conformers impossible. In the present study, an N-terminal modification of Pn3a enabled the isolation of the individual conformers, allowing activity assays to be conducted on the individual conformers using electrophysiology. The conformers were analyzed separately by nuclear magnetic resonance spectroscopy (NMR) to study their structural differences. RP-HPLC and NMR were used to study the mechanism of exchange. The later-eluting conformer was the active conformer with a rigid structure that corresponds to the published structure of Pn3a, while NMR analysis revealed the earliereluting conformer to be inactive and disordered. The exchange was found to be pH-dependent, arising in acidic solutions, possibly due to reversible disruption and formation of intramolecular salt bridges. This study reveals the nature of non-proline conformational exchange observed in Pn3a and possibly other disulfide-rich peptides, highlighting that the structure and activity of some disulfidestabilized peptides can be dramatically susceptible to disruption.

Structural Basis of the Bivalency of the TRPV1 Agonist DkTx

“…[25] It is, therefore, common to find some conformational heterogeneity in the structure of these toxins, either due to conformational exchange in the (five) dihedral angles connecting the sidechains of cysteines [26] or due to steric clashes of bulky sidechains, brought in proximity as a consequence of the covalent bonds stabilising the ICK pseudo knot. [27] Here we,…”

“…The presence of complex modes of motion is not surprising considering that in the ICK fold, the disulfide bonds (which account for >10 % of the amino acid content) are the only residues that are conserved [25] . It is, therefore, common to find some conformational heterogeneity in the structure of these toxins, either due to conformational exchange in the (five) dihedral angles connecting the sidechains of cysteines [26] or due to steric clashes of bulky sidechains, brought in proximity as a consequence of the covalent bonds stabilising the ICK pseudo knot [27] . Here we, therefore, focus our analysis on the relative changes in dynamics in the regions of the proposed stabilising tyrosine stacking interaction, in the context of the introduced mutations.…”