Unraveling C‑Peptide's Role in MIDY: A Structural Perspective

Proinsulin folding requires dynamic positioning of the C-peptide to guide A- and B-chain alignment and disulfide pairing. Mutant INS-gene-induced diabetes of youth (MIDY) arises when single-residue substitutions disrupt this process. We mapped the conformational free-energy landscapes of wild-type (WT) proinsulin and seven MIDY variants using metadynamics and molecular dynamics simulations. WT exhibits a deep free-energy minimum at compact conformations. In contrast, MIDY mutants display a continuum of destabilization: E-(A4)K retains near-WT stability, Akita (C-(A7)-Y), V-(B18)-A, and R-(Cpep + 2)C show moderate loss of the native basin, while H-(B5)-D, L-(A16)-P, and Y-(B26)C collapse the closed-open barrier and populate misfolded open states >50% of the time. Structural analyses reveal that WT and E-(A4)-K preserve robust A-C docking, with the C-peptide flexibly engaging the A-chain groove. Destabilizing mutants progressively erode these native A-C contacts while forming compensatory, non-native B-C interactions. Per-residue energy decomposition highlights the loss of canonical salt bridges and emergence of aberrant electrostatic and hydrophobic hot spots, correlating with the collapse of the folding free-energy barrier. Secondary-structure analysis further shows that mutants rigidify the normally disordered C-peptide, increasing helical or strand propensity in a mutation-specific manner. Collectively, these findings establish a continuum from near-native stability to overt misfolding, mechanistically linking single-site mutations to altered folding landscapes and aggregation risk in MIDY. The results highlight the C-peptide as a dynamic linchpin of proinsulin folding and suggest that restoring its flexible docking could provide a therapeutic avenue.