A Conserved Histidine in Insulin Is Required for the Foldability of Human Proinsulin

The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of HisB10 is well defined: although not required in the mature hormone for receptor binding, in the islet β cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of HisB5 is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an AlaB5-insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the AlaB5 analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of AlaB5 and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of HisB5 in native insulin. Substantial receptor binding activity is retained (80 ± 10% relative to the parent monomer). Although the thermodynamic stability of the AlaB5 analog is decreased (ΔΔGu = 1.7 ± 0.1 kcal/mol), consistent with loss of HisB5-related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by HisB5 facilitate alignment of CysA7 and CysB7 in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.