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

Abstract

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.