Chiral mutagenesis of insulin’s hidden receptor-binding surface: structure of an Allo-isoleucineA2 analogue

Xu, Bin; Hua, Qing; Nakagawa, Satoe H.; Jia, Wenhua; Chen, Ying; Katsoyannis, Panayotis G.; Weiss, Michael A.

doi:10.1006/jmbi.2001.5377

Cited by 45 publications

(89 citation statements)

References 48 publications

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“…Further, the activity of insulin is enhanced by chiral substitution of Phe B24 by D-Phe or D-Ala (20,50); these substitutions appear to be incompatible with native structure. Whereas the C-terminal segment of the B chain functions to contact the Nand C-terminal domains of the receptor ␣-subunit (56), its reorganization is proposed to expose an otherwise hidden functional surface in the A chain (4,54,(57)(58)(59). 6 Such models are consistent with photo-cross-linking studies of site-specific para-azido-Phe derivatives of insulin (56,59,60).…”

Section: B25mentioning

confidence: 72%

Chiral Mutagenesis of Insulin

Nakagawa

Hua

et al. 2006

Journal of Biological Chemistry

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Insulin contains a ␤-turn (residues B20 -B23) interposed between two receptor-binding elements, the central ␣-helix of the B chain (B9 -B19) and its C-terminal ␤-strand (B24 -B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive dihedral angles within a classic type-I ␤-turn. Insulin, a small globular protein secreted by pancreatic ␤ cells, plays a central role in the control of vertebrate metabolism (1). The hormone is stored in glucose-regulated secretory granules as microcrystalline arrays of Zn 2ϩ -stabilized hexamers (2) and functions as a Zn 2ϩ -free monomer. The protein contains two chains, designated A (21 residues) and B (30 residues). The structure of insulin is well characterized (3). In the T state, 3 the predominant conformation of the monomer in solution (4 -6), the A chain contains N-and C-terminal ␣-helices; the B chain contains a central ␣-helix flanked by N-and C-terminal extended segments (Fig. 1A). The product of a single-chain precursor, designated proinsulin (7), insulin contains three disulfide bridges required for its folding, stability, and function (5, 8 -10). Structure-function relationships have been extensively investigated (for review, see Ref. 11).The B chain of insulin contains two ␤-turns (boxed in Fig. 1A, see below). The first is a type-IIЈ ␤-turn comprising residues B7-B10 (black box); the second is a type-I ␤-turn comprising residues B20 -B23 (red box; ball-and-stick model in Fig. 1B). Each contains one or more conserved glycines: Gly B8 , Gly B20 , and Gly B23 (Fig. 1C). These residues exhibit positive angles ("D-glycines") and, therefore, reside on the right side of the Ramachandran plane in regions unfavorable for L-amino acids. Gly B8 , which adjoins the A7-B7 inter-chain disulfide bridge, is invariant among vertebrate insulins and highly conserved among insulin-related growth factors (black box in Fig. 1C). Mutations at B8 markedly impair the stability of insulin and the yield of chain combination (12, 13); the efficiency of recombinant expression of a single-chain precursor (mini-proinsulin) in Saccharomyces cerevisiae is likewise reduced (14,15). Whereas such unstable insulin analogs can be highly active, D-amino acid substitutions at B8 augment stability but markedly impair activity (12,13). Despite these profound stereospecific differences, the overall structures of D and L B8 analogs closely resemble native insulin (13,16).In this report we employ "chiral mutagenesis" to investigate the B20 -B23 ␤-turn. The turn sequence is notable for conserved negative and positive charges (Glu B21 and Arg B22 in eutherian mammals) flanked by glycines (Fig. 1C). Experimental design is motivated by the pattern of main-chain dihedral angles (Table 1). To investigate the interrelation of structure, activity, and stability, isomeric analogs have been synthesized in which Gly B20 or Gly B23 is substituted by D-or L-Ala; control * This work was supported i...

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Section: B25mentioning

confidence: 72%

Chiral Mutagenesis of Insulin

Nakagawa

Hua

et al. 2006

Journal of Biological Chemistry

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“…8 In the 4INS structure of the T 6 insulin hexamer, B5 main-chain dihedral angles are (Ϫ85°, 117°) in molecule 1 and (Ϫ114°, 108°) in molecule 2; values in other T 6 structures are provided as supplemental material. 9 The corresponding distance in molecule 1 is 5.0 Å; this difference between molecules 1 and 2 appears to be due to a hexamer-hexamer contact in the crystal lattice. Such hexamer-hexamer contacts appear to alter the conformation of the A9 -A11 loop in molecule 2, enabling formation of the bifurcated hydrogen bond between B5 N ⑀ H and the A9 carbonyl oxygen.…”

Section: Figure 7 Substitution Of Hismentioning

confidence: 97%

“…Synthesis of Arg B5 -insulin was performed as described (9,10); see also the supplemental materials.…”

Section: Synthesis Of Insulin Analogs-human Insulin and Aspmentioning

confidence: 99%

“…9 Whereas the guanidinium NH 2 groups of Arg B5 interact only with bound water molecules, an interchain hydrogen bond is formed within each protomer from B5 N ⑀ H (Fig. 10B, pink) to the carbonyl oxygen of Cys A7 (Fig.…”

Section: Figure 7 Substitution Of Hismentioning

confidence: 99%

“…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).…”

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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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Comparative Properties of Insulin‐like Growth Factor 1 (IGF‐1) and [Gly7D‐Ala]IGF‐1 Prepared by Total Chemical Synthesis

Sohma¹,

Pentelute²,

Whittaker³

et al. 2008

Angewandte Chemie

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Chemie für Biologen: IGF‐1 und [Gly7D‐Ala]IGF‐1 wurden durch eine chemische Totalsynthese hergestellt, und ihre Faltung und Rezeptorbindung wurden verglichen (siehe Schema). Dieser Ansatz erleichtert den Einbau ungewöhnlicher Aminosäuren, der auf biosynthetischem Weg problematisch oder unmöglich ist. Daraus ergeben sich Perspektiven für Struktur‐Aktivitäts‐Studien mit möglichen Auswirkungen auf die Krebs‐ und Diabetes‐mellitus‐Therapie.

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Chiral mutagenesis of insulin’s hidden receptor-binding surface: structure of an Allo-isoleucineA2 analogue

Cited by 45 publications

References 48 publications

Chiral Mutagenesis of Insulin

Chiral Mutagenesis of Insulin

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

Comparative Properties of Insulin‐like Growth Factor 1 (IGF‐1) and [Gly7D‐Ala]IGF‐1 Prepared by Total Chemical Synthesis

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