title: Chiral mutagenesis of insulin's hidden receptor-binding surface: structure of an allo-isoleucine(A2) analogue.
abstract: The hydrophobic core of vertebrate insulins contains an invariant isoleucine residue at position A2. Lack of variation may reflect this side-chain's dual contribution to structure and function: Ile(A2) is proposed both to stabilize the A1-A8 alpha-helix and to contribute to a "hidden" functional surface exposed on receptor binding. Substitution of Ile(A2) by alanine results in segmental unfolding of the A1-A8 alpha-helix, lower thermodynamic stability and impaired receptor binding. Such a spectrum of perturbations, although of biophysical interest, confounds interpretation of structure-activity relationships. To investigate the specific contribution of Ile(A2) to insulin's functional surface, we have employed non-standard mutagenesis: inversion of side-chain chirality in engineered monomer allo-Ile(A2)-DKP-insulin. Although the analogue retains native structure and stability, its affinity for the insulin receptor is impaired by 50-fold. Thus, whereas insulin's core readily accommodates allo-isoleucine at A2, its activity is exquisitely sensitive to chiral inversion. We propose that the Ile(A2) side-chain inserts within a chiral pocket of the receptor as part of insulin's hidden functional surface.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4935, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Xu
authors.forename: Qing-xin
authors.initials: QX
authors.lastname: Hua
authors.forename: Satoe H
authors.initials: SH
authors.lastname: Nakagawa
authors.forename: Wenhua
authors.initials: W
authors.lastname: Jia
authors.forename: Ying-Chi
authors.initials: YC
authors.lastname: Chu
authors.forename: Panayotis G
authors.initials: PG
authors.lastname: Katsoyannis
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D007328:Insulin; D007532:Isoleucine; D011972:Receptor, Insulin; D019791:Guanidine