Zinc binding, circular dichroism, and equilibrium sedimentation studies on insulin (bovine) and several of its derivatives

Goldman, Jack A.; Carpenter, Frederick H.

doi:10.1021/bi00719a015

Cited by 210 publications

(158 citation statements)

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“…As expected, our ITC experiments with human and porcine insulin gave almost identical dissociation constants of dimerization (K d ) of ϳ9 M, and the changes in Gibbs free energies of dissociation (⌬G (28), whereas this approach for porcine insulin gave K d of ϳ7 M (at pH 7.0) (44). Altogether, the dimerization dissociation constants for human and porcine insulin determined in this study fall well into the range of K d values determined experimentally by other groups and different techniques.…”

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

confidence: 83%

“…In this process, we correlate the binding affinities of the analogues to IR (determined in rat adipose membranes) using isothermal titration microcalorimetry (ITC) dilution experiments, performed to assess the thermodynamic contributions toward hormone dissociation. The wild-type insulin (capable of dimer formation) and des(B23-B30)octapeptide insulin (DOI, not able to dimerize (28,29)) were used as references for the dimeric and monomeric forms, respectively. This study is also complemented by the analysis of two novel crystal structures and two previously published analogue crystal structures that have not been hitherto discussed in the context presented here.…”

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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-supporting

confidence: 83%

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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“…The ligand-bound state is known as the R-state, and the apo-form is known as the T-state after the nomenclature of Monod et al (1965). Because the binding constant for phenolic ligands is weaker than the binding constant for zinc (Goldman & Carpenter, 1974;McGraw & Lindenbaum, 1990), it is thought that injection of a solution of R-state insulin hexamers must first convert to T-state hexamers, followed by dissociation.…”

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confidence: 99%

Physicochemical basis for the rapid time‐action of Lys^B28Pro^B29‐insulin: Dissociation of a protein‐ligand complex

et al. 1996

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The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, L y~~~~P r o~~~-h u m a n insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.

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“…Thus PTTH could form a tertiary structure with a hydrophobic core similar to all known insulins including those of the hagfish [23], the pig and the human [5,24] prevent the formation of an insulin-like fold although it might destabilise it. In mammalian insulins, removal of the A21 Asn and B30 Ala leads to changes in conformation as measured by circular dichroism [25]. These are similar to changes observed on dilution of native insulin which have been interpreted as being due to a reduction in the dimer content of an insulin solution [ 181.…”

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confidence: 82%

Prothoracicotropic hormone has an insulin‐like tertiary structure

et al. 1987

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A three-dimensional model of PTTH-II has been constructed using interactive computer graphics and energy minimisation techniques, assuming homology with porcine insulin, the structure of which has been determined by X-ray analysis. The model shows that PTTH-II can assume an insulin-like tertiary structure, which is compact with the exception of the sequence variable NH,-terminal amino acids of the B chain. Most of the hydrophobic core residues including A2 Be, A6 Cys, Al 1 Cys, Al6 Leu, A20 Cys, Bll Leu, B15 Leu and B19 Cys are identical in PTTH-II and insulins. The glycines at Al, B8 and B23 allow the chain to assume the characteristic tertiary interactions of the insulin fold and although polypeptide chains are shorter at the COOH-termini of the A and B chains and extended at the NH,-terminus of the B chain, the insulin-like tertiary structure can still be assumed. It is unlikely that PTTH-II forms either dimers or hexamers, characteristic of porcine and human insulin, and the model is consistent with the inability of PTTH-II to bind anti-insulin antibodies or insulin receptors. A hydrophobic surface region of PTTH-II may be involved in intermolecular actions of physiological relevance. We discuss the implications of our model for evolution of this family of hormones and growth factors.

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Zinc binding, circular dichroism, and equilibrium sedimentation studies on insulin (bovine) and several of its derivatives

Cited by 210 publications

References 45 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

Physicochemical basis for the rapid time‐action of Lys^B28Pro^B29‐insulin: Dissociation of a protein‐ligand complex

Prothoracicotropic hormone has an insulin‐like tertiary structure

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Zinc binding, circular dichroism, and equilibrium sedimentation studies on insulin (bovine) and several of its derivatives

Cited by 210 publications

References 45 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

Physicochemical basis for the rapid time‐action of LysB28ProB29‐insulin: Dissociation of a protein‐ligand complex

Prothoracicotropic hormone has an insulin‐like tertiary structure

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Physicochemical basis for the rapid time‐action of Lys^B28Pro^B29‐insulin: Dissociation of a protein‐ligand complex