Comparison of predicted C-peptide structures of human and venom insulin G1: A putative γ-glutamyl carboxylase recognition motif in venom insulin.

The connecting peptide (C-peptide) of human insulin has been implicated in native disulfide bond formation and diverse biological functions; however, its structural significance and potential functional roles in venom insulins remain poorly understood. The present study compares the structural significance of the C-peptide in human insulin (hum-ins) and Conus geographus venom insulin G1 (Vcon-ins G1) using computational methods. The proinsulin of both species is identical in length (86 residues); however, mature Vcon-ins G1 is seven residues shorter, while its C-peptide is correspondingly seven residues longer. VCon-ins G1 also exhibits distinctive features, including posttranslational modifications such as γ-carboxyglutamic acid (Gla), 4-trans-hydroxyproline, and C-terminal amidation, as well as a monomeric solution state and rapid activation of the human insulin receptor. Using AlphaFold 3-predicted proinsulin structures, this study investigates hydrophobicity profiles, disulfide conformations, and oxidative folding pathways. A contrasting aggregation propensity is observed: in hum-ins, the C-peptide is less aggregation-prone than the mature chain, whereas the reverse is seen in Vcon-ins G1. Disulfide conformations also differ markedly: hum-ins displays relatively confined disulfide conformations, whereas Vcon-ins G1 exhibits more diffuse and strained disulfides, with the C-peptide influencing both systems. The predicted sequential order of disulfide formation differed markedly between hum-ins and Vcon-ins G1, indicating distinct oxidative folding pathways influenced by the C-peptide. A putative γ-glutamyl carboxylase recognition motif and differential accessibility of glutamic acid residues with and without disulfide bonds suggest that the C-peptide of Vcon-ins G1 may serve as a potential recognition site for γ-glutamyl carboxylase. Comparative phylogenetic analysis of proinsulin and C-peptide sequences revealed region-specific diversification of C-peptides, potentially reflecting evolution of features that facilitate recognition by modifying enzymes such as γ-glutamyl carboxylase. These findings suggest that the C-peptide of Vcon-ins G1 has distinct structural and evolutionary roles that may contribute to its oxidative folding, posttranslational modification, and functional specialization.