title: Aromatic anchor at an invariant hormone-receptor interface: function of insulin residue B24 with application to protein design.
abstract: Crystallographic studies of insulin bound to fragments of the insulin receptor have recently defined the topography of the primary hormone-receptor interface. Here, we have investigated the role of Phe(B24), an invariant aromatic anchor at this interface and site of a human mutation causing diabetes mellitus. An extensive set of B24 substitutions has been constructed and tested for effects on receptor binding. Although aromaticity has long been considered a key requirement at this position, Met(B24) was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchor at the hormone-receptor interface. These findings motivated further substitution of Phe(B24) by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [Cha(B24)]insulin likewise exhibited high activity. Furthermore, its resistance to fibrillation and the rapid rate of hexamer disassembly, properties of potential therapeutic advantage, were enhanced. The crystal structure of the Cha(B24) analog, determined as an R6 zinc-stabilized hexamer at a resolution of 1.5 Å, closely resembles that of wild-type insulin. The nonplanar aliphatic ring exhibits two chair conformations with partial occupancies, each recapitulating the role of Phe(B24) at the dimer interface. Together, these studies have defined structural requirements of an anchor residue within the B24-binding pocket of the insulin receptor; similar molecular principles are likely to pertain to insulin-related growth factors. Our results highlight in particular the utility of nonaromatic side chains as probes of the B24 pocket and suggest that the nonstandard Cha side chain may have therapeutic utility.
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Vijay
authors.initials: V
authors.lastname: Pandyarajan
authors.affiliation: the La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Smith
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: Linda
authors.initials: L
authors.lastname: Whittaker
authors.affiliation: Physiology.
authors.forename: Gabriella P
authors.initials: GP
authors.lastname: Cox
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Nalinda
authors.initials: N
authors.lastname: Wickramasinghe
authors.affiliation: the Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia, and.
authors.forename: John G
authors.initials: JG
authors.lastname: Menting
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Zhu-li
authors.initials: ZL
authors.lastname: Wan
authors.affiliation: From the Departments of Biochemistry.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Whittaker
authors.affiliation: Physiology, Medicine, and.
authors.forename: Faramarz
authors.initials: F
authors.lastname: Ismail-Beigi
authors.affiliation: the Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia, and the Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia lawrence@wehi.edu.au.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Lawrence
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; D000818:Animals; D017124:Conserved Sequence; D015195:Drug Design; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007328:Insulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000378:metabolism; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007328:Insulin; D010649:Phenylalanine; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin
keywords: Diabetes; Hormone; Mutagenesis; Protein Design; Protein Structure; Receptor Tyrosine Kinase; nonstandard mutagenesis