4-azidophenylalanine and Diabetes-Mellitus

Mutations in human insulin cause an autosomal-dominant syndrome of diabetes and fasting hyperinsulinemia. We demonstrate by residue-specific photo cross-linking that diabetes-associated mutations occur at receptor-binding sites. The studies use para-azido-phenylalanine, introduced at five sites by total protein synthesis. Because two such sites (Val(A3) and Phe(B24)) are largely buried in crystal structures of the free hormone, their participation in receptor binding is likely to require a conformational change to expose a hidden functional surface. Our results demonstrate that this surface spans both chains of the insulin molecule and includes sites of rare human mutations that cause diabetes.

Topics: Azides; Binding Sites; Diabetes Mellitus; Humans; Insulin; Molecular Structure; Mutation; Phenylalanine; Receptor, Insulin

How insulin binds to and activates the insulin receptor has long been the subject of speculation. Of particular interest are invariant phenylalanine residues at consecutive positions in the B chain (residues B24 and B25). Sites of mutation causing diabetes mellitus, these residues occupy opposite structural environments: Phe(B25) projects from the surface of insulin, whereas Phe(B24) packs against the core. Despite these differences, site-specific cross-linking suggests that each contacts the insulin receptor. Photoactivatable derivatives of insulin containing respective p-azidophenylalanine substitutions at positions B24 and B25 were synthesized in an engineered monomer (DKP-insulin). On ultraviolet irradiation each derivative cross-links efficiently to the receptor. Packing of Phe(B24) at the receptor interface (rather than against the core of the hormone) may require a conformational change in the B chain. Sites of cross-linking in the receptor were mapped to domains by Western blot. Remarkably, whereas B25 cross-links to the C-terminal domain of the alpha subunit in accord with previous studies (Kurose, T., et al. (1994) J. Biol. Chem. 269, 29190-29197), the probe at B24 cross-links to its N-terminal domain (the L1 beta-helix). Our results demonstrate that consecutive residues in insulin contact widely separated sequences in the receptor and in turn suggest a revised interpretation of electron-microscopic images of the complex. By tethering the N- and C-terminal domains of the extracellular alpha subunit, insulin is proposed to stabilize an active conformation of the disulfide-linked transmembrane tyrosine kinase.

Topics: Amino Acid Sequence; Animals; Azides; Blotting, Western; CHO Cells; Chymotrypsin; Cricetinae; Cross-Linking Reagents; Diabetes Mellitus; Dimerization; Disulfides; Exons; Humans; Insulin; Models, Biological; Models, Molecular; Molecular Sequence Data; Mutation; Phenylalanine; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Receptor, Insulin; Sequence Homology, Amino Acid; Trypsin; Ultraviolet Rays