title: Non-standard insulin design: structure-activity relationships at the periphery of the insulin receptor.
abstract: The design of insulin analogues has emphasized stabilization or destabilization of structural elements according to established principles of protein folding. To this end, solvent-exposed side-chains extrinsic to the receptor-binding surface provide convenient sites of modification. An example is provided by an unfavorable helical C-cap (Thr(A8)) whose substitution by favorable amino acids (His(A8) or Arg(A8)) has yielded analogues of improved stability. Remarkably, these analogues also exhibit enhanced activity, suggesting that activity may correlate with stability. Here, we test this hypothesis by substitution of diaminobutyric acid (Dab(A8)), like threonine an amino acid of low helical propensity. The crystal structure of Dab(A8)-insulin is similar to those of native insulin and the related analogue Lys(A8)-insulin. Although no more stable than native insulin, the non-standard analogue is twice as active. Stability and affinity can therefore be uncoupled. To investigate alternative mechanisms by which A8 substitutions enhance activity, multiple substitutions were introduced. Surprisingly, diverse aliphatic, aromatic and polar side-chains enhance receptor binding and biological activity. Because no relationship is observed between activity and helical propensity, we propose that local interactions between the A8 side-chain and an edge of the hormone-receptor interface modulate affinity. Dab(A8)-insulin illustrates the utility of non-standard amino acids in hypothesis-driven protein design.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. weiss@biochemistry.cwru.edu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
authors.forename: Zhuli
authors.initials: Z
authors.lastname: Wan
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Ying-Chi
authors.initials: YC
authors.lastname: Chu
authors.forename: Satoe H
authors.initials: SH
authors.lastname: Nakagawa
authors.forename: G Thomson
authors.initials: GT
authors.lastname: Burke
authors.forename: Wenhua
authors.initials: W
authors.lastname: Jia
authors.forename: Rosanna
authors.initials: R
authors.lastname: Hellmich
authors.forename: Panayotis G
authors.initials: PG
authors.lastname: Katsoyannis
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000613:Aminobutyrates / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D015195:Drug Design; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000096:biosynthesis; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D000613:Aminobutyrates; D007328:Insulin; D008055:Lipids; C005959:2,4-diaminobutyric acid; D011972:Receptor, Insulin