How Insulin Binds: the B-Chain α-Helix Contacts the L1 β-Helix of the Insulin Receptor

Huang, Kun; Xu, Bin; Hu, Shiyan; Chen, Ying; Hua, Qing; Qu, Yan; Li, Biaoru; Wang, Shuhua; Wang, Run Ying; Nakagawa, Satoe H.; Theede, Anne Mette; Whittaker, Jonathan; Meyts, Pierre De; Katsoyannis, Panayotis G.; Weiss, Michael A.

doi:10.1016/j.jmb.2004.05.023

Cited by 74 publications

(96 citation statements)

References 86 publications

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“…Consistency was also obtained in assays of insulin variants containing single Ala substitutions at classical receptor contact sites resolved in the original μIR structure (SI Appendix, Fig. S2 C and D and Table S1) (2,(13)(14)(15). Together, these data imply that residues B24-B26 make similar contributions to the binding of insulin to the μIR and holoreceptor.…”

Section: Resultssupporting

confidence: 55%

“…[ 13 C, 15 N]-labeled peptides were prepared by solid-phase peptide synthesis, purified by HPLC, and assessed by MS. Unlabeled B23-B30 octapeptides containing Ala, Gly, or ΔPhe substitutions were likewise prepared; ΔPhe-containing octapeptides were also synthesized as described (12). Labeled or variant insulins were prepared by semisynthesis (42).…”

Section: Methodsmentioning

confidence: 99%

“…Labeled or variant insulins were prepared by semisynthesis (42). Analog 1 was prepared by chain combination (15). Biosynthetic isotopic labeling of the α-helical domain of analogs 2 and 3 (residues B1-B22 and A1-A21) was accomplished in Pichia pastoris (14).…”

Section: Methodsmentioning

confidence: 99%

“…Data were processed using NMRPipe (62). Structures of analogs 1 and 4 were determined as previously described (6,15); interpretation of long-range NOEs was aided by use of multiple mixing times and model-based backcalculation of predicted NOESY spectra and aromatic ring-current shifts. Heteronuclear studies of analog 2 were performed as previously described (22).…”

Section: Methodsmentioning

confidence: 99%

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Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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149

257

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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 Bchain 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-β 2 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 receptorbound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.diabetes mellitus | signal transduction | receptor tyrosine kinase | metabolism | protein structure H ow insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24-B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3-6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residuespecific photo-cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ) 2 receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1*) comprises the central β-sheet (L1-β 2 ) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical Cterminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits' intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently...

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Section: Resultssupporting

confidence: 55%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

149

257

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“…Essential residues implicated in insulin binding to the IR include Gly-1, Gln-5, Tyr-19, and Asn-21 on the A chain and Tyr-16, Gly 23, Phe-24, Phe-25, and Tyr-26 (GFFY motif) on the B chain that together form the ''classical binding surface'' (Fig. 1 A) (2,(20)(21)(22). Other important residues include IleA2 and ValA3, which are likely exposed during binding to the IR, and LeuA13, ValB12, and LeuB17 that maintain secondary structure.…”

Section: Resultsmentioning

confidence: 99%

An insulin-like peptide regulates egg maturation and metabolism in the mosquito Aedes aegypti

Brown¹,

Clark²,

Gulia³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

227

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Ingestion of vertebrate blood is essential for egg maturation and transmission of disease-causing parasites by female mosquitoes. Prior studies with the yellow fever mosquito, Aedes aegypti, indicated blood feeding stimulates egg production by triggering the release of hormones from medial neurosecretory cells in the mosquito brain. The ability of bovine insulin to stimulate a similar response further suggested this trigger is an endogenous insulin-like peptide (ILP). A. aegypti encodes eight predicted ILPs. Here, we report that synthetic ILP3 dose-dependently stimulated yolk uptake by oocytes and ecdysteroid production by the ovaries at lower concentrations than bovine insulin. ILP3 also exhibited metabolic activity by elevating carbohydrate and lipid storage. Binding studies using ovary membranes indicated that ILP3 had an IC 50 value of 5.9 nM that was poorly competed by bovine insulin. Autoradiography and immunoblotting studies suggested that ILP3 binds the mosquito insulin receptor (MIR), whereas loss-of-function experiments showed that ILP3 activity requires MIR expression. Overall, our results identify ILP3 as a critical regulator of egg production by A. aegypti.

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Analysis of Protein Ligand‐Receptor Binding by Photoaffinity Cross‐Linking

2015

CP Protein Science

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Photoaffinity cross-linking is a rapidly developing technology for studying biomolecular interactions, including protein ligand-receptor binding. This technology provides detailed binding information including receptor contact sites, active conformation of receptor-ligand complexes, global binding surfaces, and binding modes. Advancements in genetic technology have enabled non-natural photoactive amino acid derivatives to be incorporated into designer or target proteins, providing a host of new opportunities for manufacturing protein photo-probes while bypassing the traditional peptide or small protein limits of classical chemical synthesis. This unit provides several protocols for performing basic photoaffinity cross-linking and related analyses for applications in ligand-receptor binding and protein-protein interactions.

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How Insulin Binds: the B-Chain α-Helix Contacts the L1 β-Helix of the Insulin Receptor

Cited by 74 publications

References 86 publications

Protective hinge in insulin opens to enable its receptor engagement

Protective hinge in insulin opens to enable its receptor engagement

An insulin-like peptide regulates egg maturation and metabolism in the mosquito Aedes aegypti

Analysis of Protein Ligand‐Receptor Binding by Photoaffinity Cross‐Linking

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