Alanine Scanning Mutagenesis of Insulin

Kristensen, Claus; Kjeldsen, Thomas; Wiberg, Finn C.; Schäffer, Lauge; Hach, Morten; Havelund, Svend; Bass, Joseph; Steiner, Donald F.; Andersen, Asser S.

doi:10.1074/jbc.272.20.12978

Cited by 204 publications

(252 citation statements)

References 32 publications

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“…This observation rationalizes (i) the high activity of an Ala B26 analog (36), (ii) the dispensability of Tyr B26 in truncated analogs (37), and (iii) that whereas holoreceptor substitution Arg14Ala (near both Tyr B26 and the B25 main chain) impairs insulin binding >10 3 -fold, Ala substitution of Asp12 (in contact only with Tyr B26 ) impairs hormone binding by only 6-fold (33). We suggest that the conservation of Tyr B26 (2) arises not from its role in receptor binding but instead from its contribution to proinsulin folding (38) and insulin self-assembly (2,39).…”

Section: Discussionmentioning

confidence: 94%

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

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

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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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Section: Discussionmentioning

confidence: 94%

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

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

Self Cite

149

257

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“…32 Our results demonstrate for the first time that structural flexibility of the N-terminal end of the B-chain of insulin is not a prerequisite for binding and that an insulin analog not capable of forming the classical R-state is fully active. Since the first four amino acids of the B-chain do not have a large affect on insulin interaction with the insulin receptor, 42,43 the ''functional'' difference between the T-state and the R-state lies around the GlyB8 ''hinge'' region. Dihedral angles of GlyB8 range from positive in the T-state (56.4 6 4.1) to negative in the R-state (À63.0 6 3.12).…”

Section: Discussionmentioning

confidence: 99%

“…42,49,50 Shortly, the mutation to cysteine residues were introduced in selected positions in the insulin coding sequence by overlapping PCRs. The insulin precursors were expressed in Saccharomyces cerevisiae as proinsulin-like singlechains consisting of a spacer GluGluAlaGluAlaGluAlaProLys (EEAEAEAPK) followed by the B-chain (B1-B29), a mini C-peptide Ala Ala Lys (AAK) and the A-chain (A1-A21).…”

Section: Plasmids Construction and Expressionmentioning

confidence: 99%

Insulin analog with additional disulfide bond has increased stability and preserved activity

et al. 2013

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Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Ca-Ca distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the Bchain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

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“…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%

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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Alanine Scanning Mutagenesis of Insulin

Cited by 204 publications

References 32 publications

Protective hinge in insulin opens to enable its receptor engagement

Protective hinge in insulin opens to enable its receptor engagement

Insulin analog with additional disulfide bond has increased stability and preserved activity

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

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