A structural switch in a mutant insulin exposes key residues for receptor binding 1 1Edited by A. R. Fersht

Ludvigsen, Svend; Olsen, Henrik Baare; Kaarsholm, Niels C.

doi:10.1006/jmbi.1998.1801

Cited by 87 publications

(90 citation statements)

References 23 publications

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“…The comparative backbone structures of the L1 domains of IR and IGF1R generally follow each other closely owing to their high (70%) sequence identity (rmsd for C ␣ atoms of IR-1 and IGF1R is 1.2 Å; and for IR-2 and IGF1R is 1.3 Å). There is a major difference between the structures of IR residues 39-48 and the equivalent sequence in IGF1R (residues [35][36][37][38][39][40][41][42] in the second leucine-rich repeat of the L1 domain (Fig. 3), with the largest C ␣ deviations being Ͼ4 Å.…”

Section: Resultsmentioning

confidence: 99%

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

Lou¹,

Garrett²,

McKern³

et al. 2006

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

116

129

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The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1-CR-L2) of human IR at 2.3 Å resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) ␤-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more ␣-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R.crystal structure ͉ ectodomain ͉ insulin-binding site T he insulin receptor (IR), like the type-1 insulin-like growth factor receptor (IGF1R), is a member of the receptor tyrosine kinase family, and is a large, transmembrane, glycoprotein dimer consisting of several structural domains (1, 2). The N-terminal half of the ectodomain contains two leucine-rich repeat domains (L1 and L2) separated by a cys-rich region (CR) (1, 3). The C-terminal half of the IR ectodomain consists of three fibronectin type III domains, the second of which contains an insert region of Ϸ120 residues (1, 2).Although there is no high-resolution structural information available for the IR ectodomain, the three-dimensional structure is known for the first three domains (L1-CR-L2) of the closely related IGF1R (4). This structure has provided a framework to interpret previous studies on receptor chimeras, site-specific mutants, and mutants from patients with defective receptors (see refs. 1 and 2) and has guided subsequent studies on the insulin-binding site using mutational analysis (5, 6). Three regions of the ectodomain are known to be involved in low-affinity binding by the soluble IR ectodomain. These are the L1 domain, the CR region and the last 16 residues of the ␣-chain (see refs. 1 and 7). Of these, only the first two (L1 and the CR) are important determinants of ligand specificity, because IR͞IGF1R chimeras of whole receptors (8) or minireceptors (9) are little affected by swapping the regions that contained the last 16 residues of the ␣-chain.The major determinants in L1 for insulin binding specificity lie in the first 68 residues of this domain (10, 11), based on the analysis of receptor chimeras. Twelve residues in this N-terminal segment have been further confirmed as part of the ligand-binding region by site-specific mutagenesis (see Table 1). Surprisingly, nine of these 12 residues a...

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Section: Resultsmentioning

confidence: 99%

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

Lou¹,

Garrett²,

McKern³

et al. 2006

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

116

129

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“…An alternative and simpler explanation posits a one-residue shift in register between the C-terminal B-chain β-strand and site 1 surface (45). This model envisages that Phe B25 occupies the erstwhile B24-binding pocket (and likewise Tyr B26 occupies the B25 pocket), leaving a noncanonical five-residue loop between this FY motif and the B-chain α-helix.…”

Section: Discussionmentioning

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.

145

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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“…The turn conformation is independent of zinc binding and hexamer assembly (36 -38). Studies of insulin analogs have suggested that the turn is stabilized by Phe B24 (4,39,40), because the aromatic ring packs against Leu B15 and Cys…”

Section: Discussionmentioning

confidence: 99%

“…5C and Ref. 3)) by glycine leads to an altered turn structure with near-native activity (4,40). 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.…”

Section: B25mentioning

confidence: 95%

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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A structural switch in a mutant insulin exposes key residues for receptor binding 1 1Edited by A. R. Fersht

Cited by 87 publications

References 23 publications

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

Protective hinge in insulin opens to enable its receptor engagement

Chiral Mutagenesis of Insulin

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