Importance of aliphatic side-chain structure at positions 2 and 3 of the insulin A chain in insulin-receptor interactions

Nakagawa, Satoe H.; Tager, Howard S.

doi:10.1021/bi00127a023

Cited by 86 publications

(138 citation statements)

References 47 publications

(72 reference statements)

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“…other hydrophilic residues (Table 3); 2) Nakagawa et al (4,14) reported We may ask why the insulin mutants where the conserved Val residues were replaced by Thr or Ile retained relatively high receptor-binding activities and in vivo biological potencies. By comparing the side-chains of Val, Thr, and Ile, it is seen that all the three residues contain a¯-branched side-chain.…”

Section: Discussionmentioning

confidence: 99%

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Mutational Analysis of the Three Conserved Valine Residues of Insulin and a Proposal of “Isosteric Residue”

Guo¹,

Tang²,

Zhang³

et al. 2001

IUBMB Life

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Section: Discussionmentioning

confidence: 99%

“…However, this is not an absolute rule. There are some exceptions, for example, substitution of A2Ile in insulin by Thr caused a 1,000-fold decrease in receptor-binding activity whereas replacement by Leu caused only a 20-fold loss in activity (4).…”

Section: Discussionmentioning

confidence: 99%

Mutational Analysis of the Three Conserved Valine Residues of Insulin and a Proposal of “Isosteric Residue”

Guo¹,

Tang²,

Zhang³

et al. 2001

IUBMB Life

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“…However, the single-chain insulin precursor has ϳ0.1% activity of the two-chain mature molecules (22). This has been interpreted that flexibility in the C terminus of the B-chain and a free N terminus of the A-chain are required for activity (22,23). The activity (lipogenesis) of the described single chain insulin aspart precursor featuring tryptophan in the C-peptide is further decreased 10-fold to ϳ0.01% of human insulin.…”

Section: Engineering-enhanced Eukaryotic Secretion 18246mentioning

confidence: 99%

Engineering-enhanced Protein Secretory Expression in Yeast with Application to Insulin

Kjeldsen

Ludvigsen

Diers

et al. 2002

Journal of Biological Chemistry

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Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue (Asp B28 ) expressed in yeast was enhanced 5-fold by engineering a specific interaction between an aromatic amino acid in the connecting peptide and a phenol binding site in the hydrophobic core of the molecule. This insulin precursor is characterized by significantly enhanced folding stability. The improved folding properties enhanced the secretion efficiency of the insulin precursor from 10 to 50%. The precursor remains fully in vitro convertible to mature fast-acting insulin.Administration of the two-chain 51-amino acid peptide hormone insulin is life sustaining for many of the 150 million diabetic patients in the world. Classical pancreatic extraction results in 10 -15 mg of insulin per pancreas and the number of porcine or bovine pancreases to produce current requirements of insulin (in excess of 7 metric tons) is simply not available. Consequently, a substantial fraction of insulin is today produced by eukaryotic secretory expression in the yeast Saccharomyces cerevisiae. Recently, second generation fast-acting genetically engineered insulin analogues have been engineered for improved diabetes therapy. This is exemplified by the clinically relevant fast-acting insulin, which features an amino acid substitution in position 28 of the B-chain from proline to aspartic acid and is used for treatment of diabetes mellitus (1).However, many biochemical and structural properties of insulin and proinsulin have evolved in response to differential requirements of biosynthesis, processing, transport, and storage in the ␤-cells of Langerhans islets (2, 3). Importantly, insulin and insulin analogues are readily adapted for expression in yeast as single-chain proinsulin-like precursors lacking the connecting peptide in the following configuration: amino acid residues 1-29 of the B-chain connected to the A-chain by a short removable mini-C-peptide and fused to the yeast prepro-␣ factor through a single dibasic cleavage site (secretory expression of insulin in yeast and in vitro enzymatic conversion of the precursor to insulin has recently been reviewed (4, 5)). Removal of the prepro-␣ factor by endoproteolytic cleavage by the Kex2 endoprotease in a late Golgi compartment generates a singlechain insulin precursor with self-association properties and structure essentially similar to that of human two-chain insulin (6, 7).Proteins exit the lumen of the endoplasmic reticulum (ER) 1 after folding, and frequently this is a rate-limiting step in secretion (8 -10). Herein we describe an improvement in eukaryotic secretion efficiency with application to insulin, using a strategy of structure-based rational engineering under the assumption that a more stably folded precursor molecule is ...

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“…The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. Structure-function relationships have been inferred from patterns of sequence conservation (2) and extensively probed by mutagenesis (2)(3)(4)(5)(6)(7)(8)(9)(10). A variety of evidence suggests that insulin undergoes a change in conformation on binding to the insulin receptor (IR) 2 (11).…”

mentioning

confidence: 99%

“…4 Participation of His B5 in the R-specific ␣-helix ( Fig. 2, right, red circle) causes its side chain to move from the T 6 surface to pack within a trimer interface; the B5 side chain does not participate in zinc coordination or the immediate binding of phenol. The structural environment of His B5 within an R-specific trimer interface is shown in Fig.…”

mentioning

confidence: 99%

The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery

Wan

Huang

Whittaker

et al. 2008

Journal of Biological Chemistry

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The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T 6 , T 3 R 3 f , and R 6 . Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of His B5 in wild-type structures: on the T 6 surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in His B5 pK a , in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of His B5 (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of Arg B5 -insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T 6 hexamers rather than R 6 as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3 Å , closely resembles the wild-type T 6 hexamer. Whereas Arg B5 is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving His B5 in wild-type T-states. The substantial receptor-binding activity of Arg B5 -insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers.Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues). In pancreatic ␤-cells, the hormone is stored as Zn 2ϩ -stabilized hexamers, which form microcrystalline arrays within specialized secretory granules (1). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. Structure-function relationships have been inferred from patterns of sequence conservation (2) and extensively probed by mutagenesis (2-10). A variety of evidence suggests that insulin undergoes a change in conformation on binding to the insulin receptor (IR) 2 (11). A model for induced fit is provided by the TR transition, a long range allosteric reorganization of zinc insulin hexamers (12). The structural basis of the TR transition has been extensively investigated by x-ray crystallography (13-15). In this paper, we investigate the relationship between such allosteric reorganization and biological activity. Experimental design exploits the contrasting structures of classical hexamers to introduce an electrostatic block to the TR transition.The TR transition encompasses three families of zinc insulin hexamers, designated T 6 , T 3 R 3 f , and R 6 (Fig. 1). Interconversion among these families is regulated by ionic strength (13, 16...

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Importance of aliphatic side-chain structure at positions 2 and 3 of the insulin A chain in insulin-receptor interactions

Cited by 86 publications

References 47 publications

Mutational Analysis of the Three Conserved Valine Residues of Insulin and a Proposal of “Isosteric Residue”

Mutational Analysis of the Three Conserved Valine Residues of Insulin and a Proposal of “Isosteric Residue”

Engineering-enhanced Protein Secretory Expression in Yeast with Application to Insulin

The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery

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