Satoe H. Nakagawa

Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists

The C-terminal segment of the human insulin receptor α-chain (designated αCT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-cross-linking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The αCT segment is now resolved as being engaged with the central β-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is α-helical in conformation and extends 11 residues N-terminal of the classical αCT segment boundary originally defined by peptide mapping. This tandem structural element (αCT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant αCT peptides binding to an insulin minireceptor, leads to the conclusion that putative “insulin-mimetic” peptides in the literature act at least in part as mimics of the αCT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the αCT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate α-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus.

Glia maturation factor in bovine brain: Partial purification and physicochemical characterization

Glia maturation factor (GMF) is partially purified from bovine brains by the following procedure: extraction at physiologic pH, dialysis and freeze-drying of the extract, ethanol washing of the dried powder and re-extraction of the ethanol-washed residue with Tris-buffered saline, ion-exchange chromatography with DEAE Sephadex and molecular sieving with Bio-gel P-200. The partially purified protein has an apparent molecular weight of 23,000 and an isoelectric point of 4.75, and retains both morphological transforming and mitogenic activities when tested on glioblasts. Both activities are susceptible to protease digestion and heat inactivation. The procedure results in a 400-fold purification of the morphological activity and a 1400-fold purification of the mitogenic activity. Both activities are detectable when GMF is used in nanogram quantities. The possibility that both functions are expressions of the same factor and the possible role of GMF in the differential or sequential stimulation of cell growth and maturation are discussed.

Design of an Active Ultrastable Single-chain Insulin Analog SYNTHESIS, STRUCTURE, AND THERAPEUTIC IMPLICATIONS

Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 ± 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (ΔΔGu = 0.7 ± 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 ± 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts ValA3 at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 α-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.

The A-chain of Insulin Contacts the Insert Domain of the Insulin Receptor: PHOTO-CROSS-LINKING AND MUTAGENESIS OF A DIABETES-RELATED CREVICE.

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of ValA3, which projects within a crevice between the A- and B-chains. Engineered receptor α-subunits containing specific protease sites (“midi-receptors”) are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a PapB25-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and α-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (GlyA3 and SerA3) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of AbaA3 and allo-ThrA3 analogs are reduced at least 10-fold; the activity of ThrA3-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (AbaA3) or by altered polarity (allo-ThrA3 and ThrA3). Our results provide evidence that ValA3, a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the α-subunit in a hormone-receptor complex.

Decoding the Cryptic Active Conformation of a Protein by Synthetic Photoscanning INSULIN INSERTS A DETACHABLE ARM BETWEEN RECEPTOR DOMAINS

Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic β-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain β-strand (residues B24-B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the α-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of aβ-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photoscanning mutagenesis can decode the active conformation of a protein and so illuminate cryptic constraints underlying its evolution.

Toward the Active Conformation of Insulin: Stereospecific modulation of a structural switch in the B chain.

How insulin binds to the insulin receptor has long been a subject of speculation. Although the structure of the free hormone has been extensively characterized, a variety of evidence suggests that a conformational change occurs upon receptor binding. Here, we employ chiral mutagenesis, comparison of corresponding d and l amino acid substitutions, to investigate a possible switch in the B-chain. To investigate the interrelation of structure, function, and stability, isomeric analogs have been synthesized in which an invariant glycine in a β-turn (GlyB8) is replaced by d- or l-Ser. The d substitution enhances stability (ΔΔGu 0.9 kcal/mol) but impairs receptor binding by 100-fold; by contrast, the l substitution markedly impairs stability (ΔΔGu -3.0 kcal/mol) with only 2-fold reduction in receptor binding. Although the isomeric structures each retain a native-like overall fold, the l-SerB8 analog exhibits fewer helix-related and long range nuclear Overhauser effects than does the d-SerB8 analog or native monomer. Evidence for enhanced conformational fluctuations in the unstable analog is provided by its attenuated CD spectrum. The inverse relationship between stereospecific stabilization and receptor binding strongly suggests that the B7-B10 β-turn changes conformation on receptor binding.

Enhancing the activity of a protein by stereospecific unfolding. Conformational life cycle of insulin and its evolutionary origins

A central tenet of molecular biology holds that the function of a protein is mediated by its structure. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biological constraints. A model is provided by insulin, well characterized at atomic resolution by x-ray crystallography. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element. A bifunctional β-strand mediates both self-assembly (within β-cell storage vesicles) and receptor binding (in the bloodstream). This strand is anchored by an invariant side chain (PheB24); its substitution by Ala leads to an unstable but native-like analog of low activity. Substitution by d-Ala is equally destabilizing, and yet the protein diastereomer exhibits enhanced activity with segmental unfolding of the β-strand. Corresponding photoactivable derivatives (containing l- or d-para-azido-Phe) cross-link to the insulin receptor with higher d-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is associated with accelerated fibrillation, a form of aggregation-coupled misfolding associated with cellular toxicity. Conservation of PheB24, enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences.

Complementation Analysis Demonstrates That Insulin Cross-links Both α Subunits in a Truncated Insulin Receptor Dimer

The insulin receptor is a homodimer composed of two αβ half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the α subunit. However, it has not been shown whether insulin interacts with these two sites within the same α chain or whether it cross-links a site from each α subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each α subunit. Midi-hIRs containing Ala14, Ala64, or Gly714 mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with 125I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both α subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling.

Chiral mutagenesis of insulin.contribution of the B20-B23 β -turn to activity and stability

Insulin contains a β-turn (residues B20-B23) interposed between two receptor-binding elements, the central α-helix of the B chain (B9-B19) and its C-terminal β-strand (B24-B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive φ dihedral angles within a classic type-I β-turn. Because the Ramachandran conformations of GlyB20 and GlyB23 are ordinarily forbidden to l-amino acids, turn architecture may contribute to structure or function. Here, we employ “chiral mutagenesis,” comparison of corresponding d- and l-Ala substitutions, to investigate this turn. Control substitutions are introduced at GluB21, a neighboring residue exhibiting a conventional (negative) φ angle. The d- and l-Ala substitutions at B23 are associated with a marked stereospecific difference in activity. Whereas the d-AlaB23 analog retains native activity, the l analog exhibits a 20-fold decrease in receptor binding. By contrast, d- and l-AlaB20 analogs each exhibit high activity. Stereospecific differences between the thermodynamic stabilities of the analogs are nonetheless more pronounced at B20 (ΔΔGu 2.0 kcal/mole) than at B23 (ΔΔGu 0.7 kcal/mole). Control substitutions at B21 are well tolerated without significant stereospecificity. Chiral mutagenesis thus defines the complementary contributions of these conserved glycines to protein stability (GlyB20) or receptor recognition (GlyB23).

A cavity-forming mutation in insulin induces segmental unfolding of a surrounding α-helix

To investigate the cooperativity of insulins structure, a cavity‐forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, IleA2→Ala in the A1–A8 α‐helix, does not impair disulfide pairing between chains. In accord with past studies of cavity‐forming mutations in globular proteins, a decrement was observed in thermodynamic stability (ΔΔGu 0.4–1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated α‐helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1–A8 segment. The analogs solution structure is otherwise similar to that of native insulin, including the B chains supersecondary structure and a major portion of the hydrophobic core. Our results show that (1) a cavity‐forming mutation in a globular protein can lead to segmental unfolding, (2) tertiary packing of IleA2, a residue of low helical propensity, stabilizes the A1–A8 α‐helix, and (3) folding of this segment is not required for native disulfide pairing or overall structure. We discuss these results in relation to a hierarchical pathway of protein folding and misfolding. The AlaA2 analogs low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1–A8 α‐helix in receptor recognition.