The B22 Dilemma: Structural Basis for Conformational Differences in Proinsulin B-Chain Arg22 Mutants

Proinsulin has three distinct regions: the well-folded A- and B-chains and the dynamic disordered C-peptide. The highly conserved B-chain is a hotspot for diabetes-associated mutations, including the severe loss-of-function R(B22)Q mutation linked to childhood-onset diabetes. Here, we explore R(B22)’s role in proinsulin stability using AlphaFold-predicted structures and metadynamics simulations to achieve enhanced sampling of the free energy landscape. Our results show that R(B22) stabilizes proinsulin by interacting with N86. Substituting R(B22) with E or Q disrupts this interaction, increasing conformational flexibility. The R(B22)Q variant exhibits a flattened free energy landscape, favoring unfolded states. Additional substitutions, including Gly, Ala, Lys, Tyr, Asp, and Phe, destabilize proinsulin to varying extents by weakening hydrogen bonding. Disrupting the R(B22)–N86 interaction broadly reduces inter-chain contacts, raising the risk of aggregation-prone states. Given the link between R(B22) mutations and diabetes, our study provides crucial molecular insights into proinsulin instability. These findings highlight the role of key inter-domain (A-Chain–B-chain, B-Chain–C-peptide, and A-Chain–C-peptide) interactions in maintaining protein structures and the implications this has for understanding disease-associated proinsulin variants.