Structural and energetic effects of disulfide mediation on the conformational dynamics of human calcitonin

Human calcitonin (hCT) is an endogenous peptide hormone that regulates calcium homeostasis but exhibits a strong tendency toward amyloid aggregation, thereby compromising its physiological activity and limiting its clinical application in bone-related disorders. However, the molecular mechanisms underlying hCT aggregation remain elusive, particularly regarding the precise role of the conserved intramolecular C1-C7 disulfide bond in maintaining structural stability and modulating peptide folding and self-assembly. Here, we employed extensive atomistic discrete molecular dynamics (DMD) simulations to systematically investigate the conformational dynamics and aggregation behavior of hCT under oxidized (disulfide-intact, hCT_SS) and reduced (disulfide-cleaved, hCT_SH) conditions. Monomeric hCT_SS and hCT_SH predominantly adopted disordered conformations with transient helical regions and minimal β-sheet content, while disruption of the C1-C7 disulfide bond induced a helical-to-β-sheet transition near the N-terminus. Upon self-assembly, both hCT_SS and hCT_SH formed β-sheet-rich aggregates stabilized by hydrophobic and aromatic residues (17-22), whereas cleavage of the disulfide bond further promoted intra-peptide β-hairpin formation involving residues 3-13. Free-energy landscape analyses revealed that disordered states were stabilized by entropic compensation despite elevated potential energy, whereas β-sheet-rich conformations achieved low free-energy states through enthalpic stabilization. Collectively, these findings reveal the structural and energetic mechanisms underlying hCT folding and aggregation, delineate the specific role of the C1-C7 disulfide bond in modulating its conformational dynamics, and provide molecular-level insights for the rational design of aggregation-resistant hCT analogs with enhanced therapeutic potential.