Role of C-terminal B-chain residues in insulin assembly: the structure of hexameric LysB28ProB29-human insulin

Ciszak, Ewa; Beals, John M.; Frank, Bruce H.; Baker, Jeffrey C.; Carter, Nancy D; Smith, G. D.

doi:10.1016/s0969-2126(01)00195-2

Cited by 182 publications

(189 citation statements)

References 19 publications

(36 reference statements)

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“…Its monomeric behavior did not also prohibit its crystallization as the T 3 R 3 f hexamer in the presence of zinc and phenol (16). The [NMePheB26]-insulin was an additional step in probing of the resistance of the dimer interface by prevention of the "central" B26NH-COB24 hydrogen bond of the dimer interface, combined with the loss of B26 phenolic character.…”

Section: Impact Of Modifications On Binding Affinity Of Analogues-mentioning

confidence: 99%

“…Therefore, the dissociation of insulin dimers into monomers in the bloodstream is a dilution-driven phenomenon. The clinically used and fast acting [LysB28,ProB29]-insulin (Humalog) (58) or [AspB28]-insulin (Novolog) (59,60) are not able to form dimers, but still associate into hexamers (16), providing evidence that the disruption of insulin dimerization has a fundamental impact on the endogenous action of this hormone.…”

Section: Impact Of Modifications On Binding Affinity Of Analogues-mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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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Section: Impact Of Modifications On Binding Affinity Of Analogues-mentioning

confidence: 99%

Section: Impact Of Modifications On Binding Affinity Of Analogues-mentioning

confidence: 99%

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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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“…A survey of the literature showed that the presence of organic solvents such as phenol can promote crystallization of small disulfidebonded peptides or small molecules. Porcine and human insulin (Whittingham et al, 1999;Ciszak et al, 1995) and-dendrotoxin (Skarżyń ski, 1991) are examples where application of these additives was successful. Therefore, in the next round of trials phenol was added to a final concentration of 100-200 mM in both the drop and well solutions.…”

Section: Resultsmentioning

confidence: 99%

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 fromPseudonaja textilis textilis

et al. 2006

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Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an antibleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 Å resolution, while crystals of free rTxln-1 diffracted to 1.63 Å resolution.

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“…The magnitude of the d-d band in KP-insulin (gray line in Fig. 5A) was slightly attenuated relative to the WT hexamer (black line), presumed to represent a small shift in the conformational equilibrium from R 6 to T 3 R f 3 (37,38,45). The spectrum of the parent Orn B29 -insulin (red line in Fig.…”

Section: Sec Studies Provided Evidence Of Decreased or Aberrantmentioning

confidence: 98%

Contribution of TyrB26 to the Function and Stability of Insulin

Pandyarajan¹,

Phillips²,

Rege³

et al. 2016

Journal of Biological Chemistry

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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, nonaromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakestbinding 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.Insulin, a small globular protein critical to the maintenance of metabolic homeostasis (1), provides a classical model for studies of protein structure and self-assembly (2) with longstanding application to human therapeutics (3). Insulin contains two polypeptide chains, designated A and B, that are linked by two disulfide bridges (cystines A7-B7 and A20-B19); the A chain is further stabilized by an intrachain bridge (cystine A6-A11). In pancreatic ␤-cells, insulin is stored within glucoseregulated secretory granules as zinc-coordinated hexamers (Fig. 1A). The hormone binds to a receptor-tyrosine kinase, designated the insulin receptor (IR). 5 The product of a single gene, the IR precursor is processed in the trans-Golgi network into its final disulfide-linked (␣␤) 2 homodimer conformation. The extracellular ␣ subunit contains hormone-binding elements, whereas the transmembrane ␤ subunit contains the intracellular tyrosine kinase domain (4). Alternative splicing leads to two receptor isoforms, IR-A and IR-B (5). The threedimensional structure of the holoreceptor has been visualized only at low resolution (Ͼ20 Å), but these findings have been inconclusive (for review, see Ref. 6). How extracellular binding of insulin alters the structure of the receptor, leading in turn to activation of the intracellular tyrosine kinase domains, is a major unsolved problem (7).Dissection of the IR into discrete domains has enabled crystallographic analysis of its parts. The relevant structural biology is as follows. (i) Structures of the intracellular tyrosine kinase domains at 1.9 Å resolution have been o...

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Role of C-terminal B-chain residues in insulin assembly: the structure of hexameric LysB28ProB29-human insulin

Cited by 182 publications

References 19 publications

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

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

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 fromPseudonaja textilis textilis

Contribution of TyrB26 to the Function and Stability of Insulin

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