Molecular and structural dissection of Psalmopoeus pulcher venom reveals cysteine-rich peptides that target acid-sensing ion channel 1a.

Spider venoms are rich sources of peptide toxins that modulate ion channels and receptors. Although individual toxins from the genus Psalmopoeus have been shown to be critical for probing the function of certain ion channels, the overall venom composition of this genus remains poorly characterized. In this study, we investigated the molecular and structural features of peptide toxins from the spider Psalmopoeus pulcher, focusing on peptide diversity and structural architecture. The molecular diversity of P. pulcher venom was examined through mass spectrometric analysis combined with construction of a venom-gland cDNA library. A total of 495 high-quality expressed sequence tags were obtained, including 357 toxin-like expressed sequence tags, encoding 69 nonredundant toxin precursors containing signal peptides. Based on sequence homology and cysteine frameworks, these precursors were classified into 11 families. Structural predictions using AlphaFold 3 revealed marked diversity in cysteine connectivity and folding patterns among the predicted mature peptides, encompassing canonical inhibitory cystine knot scaffolds, disulfide-directed hairpin motifs, peptides with expanded disulfide networks, and a subset of precursors predicted to adopt complex, multidomain architectures. Functional exploration combining sequence analysis, molecular docking, and limited experimental validation identified Pp1a, a psalmotoxin-1-like peptide encoded by multiple precursors, as an inhibitor of acid-sensing ion channel 1a. Collectively, this study expands current knowledge of the molecular diversity and structural characteristics of P. pulcher venom peptide and provides a foundation for further functional exploration and utilization of this venom-derived peptide resource.