X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule

Derewenda, Urszula; Derewenda, Zygmunt S.; Dodson, E.J.; Dodson, G.G.; Xiao, Bing; Markussen, Jan

doi:10.1016/0022-2836(91)90022-x

Cited by 210 publications

(268 citation statements)

References 16 publications

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“…In fact, amino-acid substitutions within these regions have been identified in the insulins from © 1997 International Union of Crystallography Printed in Great Britain -all rights reserved three individuals in whom genetic mutations resulted in the secretion of abnormal hormones associated with diabetes (Tager et al, 1979(Tager et al, , 1980Shoelson, Fickova et al, 1983;Haneda, Chan, Kowk, Rubenstein & Steiner, 1983), insulin Chicago (B25-Phe--~B25-Leu), insulin Los Angeles (B24-Phe--->B24-Ser), and insulin Wakayama (A3-Val--->A3-Leu). The structural analysis of a A1-B29 cross-linked insulin, which was completely inactive, indicates that its conformation is nearly same as that of native insulin (Derewenda et al, 1991). The lower binding potency of the cross-linked insulin results from the restraining effect of the cross-link that impedes the conformational changes necessary for generation of the binding conformation.…”

Section: Introductionmentioning

confidence: 99%

Structure of Monomeric Porcine DesB1–B2 Despentapeptide (B26–B30) Insulin at 1.65 Å Resolution

et al. 1997

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Insulin has a concentration of 10-8-10 -11M in the blood which ensures that it circulates and exerts its physiological functions in vivo as a monomer. The crystal structure of monomeric porcine desB1-B2 despentapeptide (B26-B30) insulin (DesB1-2 DPI) with M r = 4934 Da has been determined at 1.65 A resolution using the molecular replacement method. A structural comparison between DesB1-2 DPI and 2Zn insulin reveals that the conformation of DesB1-2 DPI is more similar to molecule I than molecule II of 2Zn insulin. The remarkable conformational difference between B25-Phe in DesB1-2 DPI and B25-Phe in despentapeptide (B26-B30) insulin (DPI) indicates that the residue B25-Phe possesses great flexibility and mobility.

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Section: Introductionmentioning

confidence: 99%

Structure of Monomeric Porcine DesB1–B2 Despentapeptide (B26–B30) Insulin at 1.65 Å Resolution

et al. 1997

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“…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)(4)(5)(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).…”

mentioning

confidence: 99%

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

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

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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“…However, despite decades of intensive research, many questions about the structure of insulin and its mechanism of action remain. The solid state-based structural insight into the insulin molecule is limited to inactive dimeric or hexameric storage forms (1)(2)(3), whereas the insulin monomer represents the active form of the hormone when binding to the insulin receptor (IR). 3 It is also widely accepted that insulin undergoes a profound structural change during this process (4 -6), a hypothesis supported by a plethora of highly dynamic hormone conformers identified by NMR studies (7)(8)(9)(10)(11)(12)(13).…”

mentioning

confidence: 99%

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

Antolíková

Žáková

Turkenburg

et al. 2011

Journal of Biological Chemistry

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Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Insulin is an important polypeptide hormone that controls a wide range of cellular processes such as the regulation of blood glucose uptake and has a large impact on protein and lipid metabolism. However, despite decades of intensive research, many questions about the structure of insulin and its mechanism of action remain. The solid state-based structural insight into the insulin molecule is limited to inactive dimeric or hexameric storage forms (1-3), whereas the insulin monomer represents the active form of the hormone when binding to the insulin receptor (IR).3 It is also widely accepted that insulin undergoes a profound structural change during this process (4 -6), a hypothesis supported by a plethora of highly dynamic hormone conformers identified by NMR studies (7-13). Attempts to determine the structure of the insulin-IR complex have been unsuccessful so far. However, the regions of the insulin molecule responsible for the interaction with the IR (3, 14) or for its dimerization and hexamerization (15, 16) have been functionally and structurally identified in a number of insulin analogues.The insulin molecule consists of two peptide chains, a 21-amino acid A-chain and a 30-amino acid B-chain, interconnected by two interchain and one intrachain disulfide bridges. The C terminus of the B-chain of insulin, particularly residues B24 -B26, plays a substantial role in the initial contact with the receptor. It is believed that the C terminus of the B-chain of insulin must be detached away from the central B-chain ␣-helix of insulin (2, 6). One of the main signatures of this so-called "active form" of insulin should be the exposure of the previously hidden amino acids Gly-A1, Ile-A2, and Val-A3, which are important for the interaction with IR (3). Recently, we described crystal structures of several shortened and full-length insulin analogues with modifications at the B26 position (17). The structural convergence of some of these highly active analogues (200 -400%) enabled us to postulate that the active form of human insulin is characterized by a formation of a new type II ␤-turn at positions B24 -B26.Besides its role in IR activation and IR negative cooperativity (18,19), the C terminus of the B-chain is also responsible for the formation and stabilization of the insulin dimers t...

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X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule

Cited by 210 publications

References 16 publications

Structure of Monomeric Porcine DesB1–B2 Despentapeptide (B26–B30) Insulin at 1.65 Å Resolution

Structure of Monomeric Porcine DesB1–B2 Despentapeptide (B26–B30) Insulin at 1.65 Å Resolution

Protective hinge in insulin opens to enable its receptor engagement

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

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