Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the ␣-helix B8 -B19 to the ␤-turn B20 -B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.Insulin mediates its effects by binding to the insulin receptor in the plasma membrane of target cells. The molecular mechanisms for insulin interaction with the receptor are not fully understood. The crystal structure of the insulin molecule has been known for more than 25 years (1), but it remains an open question whether the structure of insulin that binds to the receptor is similar to the crystal structure. Until the structure of bound insulin and the side chains that are actually involved in binding is identified by co-crystallization of the receptor and ligand, more information about the binding domain on insulin can be obtained using mutational approaches.The binding domain of the insulin molecule has been studied by investigating receptor binding of a number of insulins from different animal species as well as chemically modified and more recently genetically engineered insulins (2-4). These studies have provided experimental support for a model in which invariant residues from both A and B chains form a surface that binds to the insulin receptor. The putative binding domain comprises a number of residues overlapping the dimerforming surface (ValB12, TyrB16, GlyB23, PheB24, PheB25, TyrB26, GlyA1, GlnA5, TyrA19, and AsnA21) and some of the residues buried beneath the COOH terminus of the B-chain (IleA2, ValA3, GluA4) (2). Cross-linking studies with an azidophenylalanine-substituted analog have shown that one of these residues, PheB25, comes into close proximity to the insulin receptor upon binding (5).Recently, a second binding site has been proposed, involving residues LeuA13 and LeuB17 (6, 7). A biphasic binding reaction involving this second binding site could explain the negative cooperativity phenomen...

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1. To investigate the structure/function relationship of the interaction between ligand and receptor in the insulin-like growth factor I (IGF-I) and insulin receptor systems we have prepared and characterized a single-chain insulin/IGF-I hybrid. The single-chain hybrid consists of the insulin molecule combined with the C domain of IGF-I. The single-chain hybrid was found to bind with high affinity to both truncated soluble insulin receptors and membrane-bound holoreceptors. The affinity for interacting with the soluble truncated insulin receptors was 55-94% relative to insulin, and affinity for membrane-bound insulin receptors was 113% of that of insulin. Furthermore we found that the affinity of the single-chain hybrid molecule for IGF-I receptors was 19-28% relative to IGF-I. 2. The affinity of the single-chain hybrid for chimeric insulin/IGF-I receptors exceeded that of either natural ligand. This indicates that coordinately changing domains of the receptors and the ligands can induce higher affinity of ligand for receptor, supporting the idea that these receptors have a common ligand-binding site [Kjeldsen, Andersen, Wiberg, Rasmussen, Schäffer, Balschmidt, Møller and Møller (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408]. 3. In contrast with what was generally assumed about the ligand structure required for binding to the insulin receptor we demonstrate the first single-chain insulin analogue that can bind with high affinity to the insulin receptor.

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