title: Contribution of TyrB26 to the Function and Stability of Insulin: STRUCTURE-ACTIVITY RELATIONSHIPS AT A CONSERVED HORMONE-RECEPTOR INTERFACE.
abstract: Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of Tyr(B26), a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the Glu(B26) analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although Tyr(B26) is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences.
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Vijay
authors.initials: V
authors.lastname: Pandyarajan
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Nelson B
authors.initials: NB
authors.lastname: Phillips
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Nischay
authors.initials: N
authors.lastname: Rege
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia, Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Lawrence
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Whittaker
authors.affiliation: From the Departments of Biochemistry, Medicine, and Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, michael.weiss@case.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D006801:Humans; D006860:Hydrogen Bonding; D007328:Insulin / Q000737:chemistry*; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D011972:Receptor, Insulin / Q000737:chemistry; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry
chemical_list: D007328:Insulin; D021122:Protein Subunits; D014443:Tyrosine; D011972:Receptor, Insulin
keywords: diabetes; hormone; non-standard mutagenesis; protein structure; receptor-tyrosine kinase