Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists

The C-terminal segment of the human insulin receptor alpha-chain (designated alpha CT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-crosslinking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The alpha CT segment is now resolved as being engaged with the central beta-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is alpha-helical in conformation and extends 11 residues N-terminal of the classical alpha CTsegment boundary originally defined by peptide mapping. This tandem structural element (alpha CT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant alpha CT peptides binding to an insulin minireceptor, leads to the conclusion that putative "insulin-mimetic" peptides in the literature act at least in part as mimics of the alpha CT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the alpha CT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate alpha-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus.