Aromatic anchor at an invariant hormone-receptor interface: function of insulin residue B24 with application to protein design.

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.

Protective hinge in insulin opens to enable its receptor engagement.

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT).

How insulin engages its primary binding site on the insulin receptor.

Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer's disease; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin-insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein.

α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase.

The primary hormone-binding surface of the insulin receptor spans one face of the N-terminal β-helix of the α-subunit (the L1 domain) and an α-helix in its C-terminal segment (αCT). Crystallographic analysis of the free ectodomain has defined a contiguous dimer-related motif in which the αCT α-helix packs against L1 β-strands 2 and 3. To relate structure to function, we exploited expanded genetic-code technology to insert photo-activatable probes at key sites in L1 and αCT.

Supramolecular protein engineering: design of zinc-stapled insulin hexamers as a long acting depot.

Bottom-up control of supramolecular protein assembly can provide a therapeutic nanobiotechnology. We demonstrate that the pharmacological properties of insulin can be enhanced by design of "zinc staples" between hexamers. Paired (i, i+4) His substitutions were introduced at an alpha-helical surface.

Contribution of residue B5 to the folding and function of insulin and IGF-I: constraints and fine-tuning in the evolution of a protein family.

Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6-A11, A7-B7, and A20-B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7-A11, A6-B7, and A20-B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing.

Design of an insulin analog with enhanced receptor binding selectivity: rationale, structure, and therapeutic implications.

Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR.

High-affinity insulin binding: insulin interacts with two receptor ligand binding sites.

The interaction of insulin with its receptor is complex. Kinetic and equilibrium binding studies suggest coexistence of high- and low-affinity binding sites or negative cooperativity. These phenomena and high-affinity interactions are dependent on the dimeric structure of the receptor.

Characterization of a second ligand binding site of the insulin receptor.

Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the alpha subunit contain one binding site.

Rescue of ligand binding of a mutant IGF-I receptor by complementation.

The IGF-I receptor binds IGF-I with complex kinetics characterized by a curvilinear Scatchard plot, suggesting receptor heterogeneity and apparent negative cooperativity. To explore the molecular mechanisms underlying these properties, we have characterized the binding of a hybrid receptor formed from a wild-type receptor monomer and a mutant receptor monomer devoid of binding activity. Receptor hybrids were generated by transient co-transfection of cDNAs encoding wild-type and mutant receptors with unique epitope tags.

Characterization of the functional insulin binding epitopes of the full-length insulin receptor.

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.

Mapping of the insulin-like growth factor II binding site of the Type I insulin-like growth factor receptor by alanine scanning mutagenesis.

The Type I insulin-like growth factor receptor is a physiological receptor for insulin-like growth factor II (IGF-II). To characterize the molecular basis of the receptor's ligand binding properties, we have examined the effects of alanine mutations of residues in the ligand binding site of the receptor on its affinity for IGF-II. The functional epitope for IGF-II comprises residues in the N-terminal L1 domain and residues at the C-terminus of the alpha subunit.