alpha-chymotrypsin and 4-azidophenylalanine

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