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

Numerous spider venom-derived gating modifier toxinsexhibit conformationalheterogeneity during purification by reversed-phase high-performanceliquid chromatography (RP-HPLC). This conformational exchange is especiallypeculiar for peptides containing an inhibitor cystine knot motif,which confers excellent structural stability under conditions thatare not conducive to disulfide shuffling. This phenomenon is oftenattributed to proline cis/trans isomerizationbut has also been observed in peptides that do not contain a prolineresidue. Pn3a is one such peptide forming two chromatographicallydistinguishable peaks that readily interconvert following the purificationof either conformer. The nature of this exchange was previously uncharacterizeddue to the fast rate of conversion in solution, making isolation ofthe conformers impossible. In the present study, an N-terminal modificationof Pn3a enabled the isolation of the individual conformers, allowingactivity assays to be conducted on the individual conformers usingelectrophysiology. The conformers were analyzed separately by nuclearmagnetic resonance spectroscopy (NMR) to study their structural differences.RP-HPLC and NMR were used to study the mechanism of exchange. Thelater-eluting conformer was the active conformer with a rigid structurethat corresponds to the published structure of Pn3a, while NMR analysisrevealed the earlier-eluting 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 intramolecularsalt bridges. This study reveals the nature of non-proline conformationalexchange observed in Pn3a and possibly other disulfide-rich peptides,highlighting that the structure and activity of some disulfide-stabilizedpeptides can be dramatically susceptible to disruption.

Automated summary

Multiple spider venom-derived gating modifier toxins exhibit conformational heterogeneity during purification by RP-HPLC. This phenomenon, often attributed to proline cis/trans isomerization, has also been observed in peptides without a proline residue. Pn3a is one such peptide forming two distinguishable peaks that readily interconvert. In this study, an N-terminal modification of Pn3a allowed the isolation of the conformers and revealed that the earlier-eluting conformer is inactive and disordered, while the later-eluting conformer is active with a rigid structure corresponding to the published structure of Pn3a. The exchange is pH-dependent and may be caused by reversible disruption and formation of intramolecular salt bridges.