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  • Whittaker, Jonathan  (13)
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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 10 July 2012, Vol.109(28), pp.11166-71
    Description: 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. The pattern of αCT-mediated photo-cross-linking within the free and bound receptor is in accord with the crystal structure and prior mutagenesis. Surprisingly, L1 photo-probes in β-strands 2 and 3, predicted to be shielded by αCT, efficiently cross-link to insulin. Furthermore, anomalous mutations were identified on neighboring surfaces of αCT and insulin that impair hormone-dependent activation of the intracellular receptor tyrosine kinase (contained within the transmembrane β-subunit) disproportionately to their effects on insulin binding. Taken together, these results suggest that αCT, in addition to its hormone-recognition role, provides a signaling element in the mechanism of receptor activation.
    Keywords: Protein-Tyrosine Kinases -- Chemistry ; Receptor, Insulin -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: The Journal of biological chemistry, 30 December 2016, Vol.291(53), pp.27023-27041
    Description: Insulin, a protein critical for metabolic homeostasis, provides a classical model for protein design with application to human health. Recent efforts to improve its pharmaceutical formulation demonstrated that iodination of a conserved tyrosine (Tyr) enhances key properties of a rapid-acting clinical analog. Moreover, the broad utility of halogens in medicinal chemistry has motivated the use of hybrid quantum- and molecular-mechanical methods to study proteins. Here, we (i) undertook quantitative atomistic simulations of 3-[iodo-Tyr]insulin to predict its structural features, and (ii) tested these predictions by X-ray crystallography. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog, as a dimer and hexamer, exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Aromatic rings (Tyr, Phe, Phe, 3-I-Tyr, and their symmetry-related mates) at this interface adjust to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of a 3-[iodo-Tyr]insulin analog (determined as an R zinc hexamer). Given that residues B24-B30 detach from the core on receptor binding, the environment of 3-I-Tyr in a receptor complex must differ from that in the free hormone. Based on the recent structure of a "micro-receptor" complex, we predict that 3-I-Tyr engages the receptor via directional halogen bonding and halogen-directed hydrogen bonding as follows: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient σ-hole and electronegative equatorial band. Inspired by quantum chemistry and molecular dynamics, such "halogen engineering" promises to extend principles of medicinal chemistry to proteins.
    Keywords: Diabetes ; Diabetes Mellitus ; Hormone ; Molecular Dynamics ; Non-Standard Mutagenesis ; Quantum Chemistry ; Quantum Mechanics ; Weakly Polar ; Chemistry, Pharmaceutical ; Insulin -- Analogs & Derivatives ; Receptor, Insulin -- Metabolism
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 3
    Language: English
    In: The Journal of biological chemistry, 17 June 2016, Vol.291(25), pp.12978-90
    Description: 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.
    Keywords: Diabetes ; Hormone ; Non-Standard Mutagenesis ; Protein Structure ; Receptor-Tyrosine Kinase ; Insulin -- Chemistry
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 4
    Language: English
    In: Biophysical Journal, 31 January 2012, Vol.102(3), pp.621a-622a
    Keywords: Biology
    ISSN: 0006-3495
    E-ISSN: 1542-0086
    Source: ScienceDirect Journals (Elsevier)
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  • 5
    Language: English
    In: The Journal of biological chemistry, 05 January 2018, Vol.293(1), pp.47-68
    Description: Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling , of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (Δ 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for 〉140 days at 45 °C and 〉48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation is compatible with native duration of activity Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.
    Keywords: Diabetes ; Hormone ; Protein Engineering ; Protein Structure ; Receptor Tyrosine Kinase ; Hypoglycemic Agents -- Chemistry ; Insulin -- Analogs & Derivatives
    E-ISSN: 1083-351X
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  • 6
    Language: English
    In: The Journal of biological chemistry, 12 December 2014, Vol.289(50), pp.34709-27
    Description: 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.
    Keywords: Diabetes ; Hormone ; Mutagenesis ; Protein Design ; Protein Structure ; Receptor Tyrosine Kinase ; Nonstandard Mutagenesis ; Drug Design ; Phenylalanine ; Insulin -- Chemistry ; Receptor, Insulin -- Metabolism
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 7
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 19 August 2014, Vol.111(33), pp.E3395-404
    Description: 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). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe(B24) to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1-β2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile(A2), Val(A3), Val(B12), Phe(B24), and Phe(B25)) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.
    Keywords: Diabetes Mellitus ; Metabolism ; Protein Structure ; Receptor Tyrosine Kinase ; Signal Transduction ; Insulin -- Metabolism ; Receptor, Insulin -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 8
    Language: English
    In: The Journal of biological chemistry, 16 April 2010, Vol.285(16), pp.11755-9
    Description: 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. The crystal structure contains both classical axial zinc ions and novel zinc ions at hexamer-hexamer interfaces. Although soluble at pH 4, the combined electrostatic effects of the substitutions and bridging zinc ions cause isoelectric precipitation at neutral pH. Following subcutaneous injection in a diabetic rat, the analog effected glycemic control with a time course similar to that of long acting formulation Lantus. Relative to Lantus, however, the analog discriminates at least 30-fold more stringently between the insulin receptor and mitogenic insulin-like growth factor receptor. Because aberrant mitogenic signaling may be associated with elevated cancer risk, such enhanced specificity may improve safety. Zinc stapling provides a general strategy to modify the pharmacokinetic and biological properties of a subcutaneous protein depot.
    Keywords: Insulin -- Analogs & Derivatives
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 9
    Language: English
    In: The Journal of biological chemistry, 12 February 2010, Vol.285(7), pp.5040-55
    Description: 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. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His(B5) --〉 Thr markedly destabilizes the hormone (DeltaDeltaG(u) 2.0 +/- 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution Thr(B5) --〉 His (residue 4) specifies a unique structure with native (1)H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, His(B5)-IGF-I retains canonical pairing. Chemical denaturation studies indicate that His(B5) does not significantly enhance thermodynamic stability (DeltaDeltaG(u) 0.2 +/- 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of Thr(B5)-insulin is decreased 5-fold, His(B5)-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of Thr(B5) in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of His(B5) in insulin highlights its critical role in insulin biosynthesis.
    Keywords: Insulin -- Chemistry ; Insulin-Like Growth Factor I -- Chemistry
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 10
    Language: English
    In: Journal of diabetes science and technology, 01 March 2012, Vol.6(2), pp.277-88
    Description: Insulin is susceptible to thermal fibrillation, a misfolding process that leads to nonnative cross-β assembly analogous to pathological amyloid deposition. Pharmaceutical formulations are ordinarily protected from such degradation by sequestration of the susceptible monomer within native protein assemblies. With respect to the safety and efficacy of insulin pumps, however, this strategy imposes an intrinsic trade-off between pharmacokinetic goals (rapid absorption and clearance) and the requisite physical properties of a formulation (prolonged shelf life and stability within the reservoir). Available rapid-acting formulations are suboptimal in both respects; susceptibility to fibrillation is exacerbated even as absorption is delayed relative to the ideal specifications of a closed-loop system. To circumvent this molecular trade-off, we exploited structural models of insulin fibrils and amyloidogenic intermediates to define an alternative protective mechanism. Single-chain insulin (SCI) analogs were shown to be refractory to thermal fibrillation with maintenance of biological activity for more than 3 months under conditions that promote the rapid fibrillation and inactivation of insulin. The essential idea exploits an intrinsic incompatibility between SCI topology and the geometry of cross-β assembly. A peptide tether was thus interposed between the A- and B-chains whose length was (a) sufficiently long to provide the "play" needed for induced fit of the hormone on receptor binding and yet (b) sufficiently short to impose a topological barrier to fibrillation. Our findings suggest that ultrastable monomeric SCI analogs may be formulated without protective self-assembly and so permit simultaneous optimization of pharmacokinetics and reservoir life.
    Keywords: Hot Temperature ; Insulin Infusion Systems ; Hypoglycemic Agents -- Chemistry ; Proinsulin -- Chemistry
    E-ISSN: 1932-2968
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