title: Importance of aliphatic side-chain structure at positions 2 and 3 of the insulin A chain in insulin-receptor interactions.
abstract: In order to evaluate the cause of the greatly decreased receptor-binding potency of the naturally occurring mutant human insulin Insulin Wakayama ([LeuA3]insulin, 0.2% relative potency), we examined (by the semisynthesis of insulin analogues based on N alpha-PheB1,N epsilon-LysB29-bisacetyl-insulin) the importance of aliphatic side chain structure at positions A2 and A3 (Ile and Val, respectively) in directing the interaction of insulin with its receptor. Analogues bearing glycine, alanine, alpha-amino-n-butyric acid, norvaline, norleucine, valine, isoleucine, allo-isoleucine, threonine, tert-leucine, or leucine at positions A2 or A3 were assayed for their potencies in competing for the binding of 125I-labeled insulin to isolated canine hepatocytes, as were analogues bearing deletions from the A-chain amino terminus or the B-chain carboxyl terminus. Selected analogues were also analyzed by far-UV CD and absorption spectroscopy of Co2+ complexes. Our results identify that (a) Ile and Val serve well at position A2, whereas residues with other side chains (including those with straight chains, alternatively configured beta-branches, or a gamma-branch) exhibit relative receptor-binding potencies in the range 1-5%; (b) greater flexibility is allowed side-chain structure at position A3, with Ile, allo-Ile, alpha-amino-n-butyric acid, and tert-Leu exhibiting relative receptor-binding potencies in the range 11-36%; and (c) simultaneous replacements at positions A2 and A3, and deletions of the COOH-terminal domain of the insulin B chain in related analogues, yield cumulative effects. These findings are discussed with respect to a model for insulin-receptor interactions that involves a structure-orienting role for residue A2, the direct interaction of residue A3 with receptor, and multiple separately defined elements of structure and of conformational adjustment.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
authors.forename: S H
authors.initials: SH
authors.lastname: Nakagawa
authors.forename: H S
authors.initials: HS
authors.lastname: Tager
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004285:Dogs; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000737:chemistry*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013552:Swine
chemical_list: D007328:Insulin; D011972:Receptor, Insulin