Rational steering of insulin binding specificity by intra-chain chemical crosslinking

Viková, Jitka; Collinsová, Michaela; Kletvíková, Emília; Budêšínský, Miloś; Kaplan, Vojtěch; Žáková, Lenka; Veverka, Václav; Hexnerova, R.; Aviñó, Roberto J. Tarazona; Straková, Jana; Selicharová, Irena; Vaněk, Václav; Wright, D.W.; Watson, Christopher J.; Turkenburg, J.P.; Brzozowski, A.M.; Jiráček, Jiřı́

doi:10.1038/srep19431

Cited by 20 publications

(32 citation statements)

References 56 publications

(60 reference statements)

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“…It can be expected that a similar orientation will be adopted by the B24–B29 segments of the analogs discussed above (23, 47) (Figure 5). If the modifications at the B24–B29 chain of analogs provide some specific and favorable contacts with αCT-B, then this 12-amino acid segment should be oriented on the L1 domain in a similar direction to enable contacts of both peptide chains.…”

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confidence: 88%

Structural Perspectives of Insulin Receptor Isoform-Selective Insulin Analogs

Jiráček

Žáková

2017

Front. Endocrinol.

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A significant drawback of the exogenous administration of insulin to diabetics is the non-physiological profile of insulin action resulting in the insufficient suppression of hepatic glucose production, which is the main contributing factor to diabetic hyperglycemia under fasting conditions and the basis of the challenge to restore a more physiological glucose profile in diabetes. The insulin receptor (IR) exists in two alternatively spliced variants, IR-A and IR-B, with different tissue distribution. While peripheral tissues contain different proportions of both isoforms, hepatic cells almost exclusively contain IR-B. In this respect, IR-B-selective insulin analogs would be of great interest for their potential to restore more natural metabolic homeostasis in diabetes. Recent advances in the structural biology of insulin and IR have provided new clues for understanding the interaction of both proteins. This article discusses and offers some structural perspectives for the design of specific insulin analogs with a preferential binding to IR-B.

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confidence: 88%

Structural Perspectives of Insulin Receptor Isoform-Selective Insulin Analogs

Jiráček

Žáková

2017

Front. Endocrinol.

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“…Receptor binding studies with the insulin receptor in membranes of mouse embryonic fibroblasts derived from IGF-I receptor knock-out mice that solely expressed the human IR-B isoform were performed as described in detail previously ( 86 , 87 ). Binding data were analyzed, and the dissociation constant ( K d ) was determined with GraphPad Prism 5 software using a method of non-linear regression and a one-site fitting program and taking into account potential depletion of free ligand.…”

Section: Methodsmentioning

confidence: 99%

“…Receptor binding studies with the IGF-I receptor in membranes of mouse embryonic fibroblasts derived from IGF-1R knock-out mice and transfected with human IGF-1R were performed as described previously ( 86 , 87 ). Binding data were analyzed, and the dissociation constants were determined and calculated by the same method as for IR-B.…”

Section: Methodsmentioning

confidence: 99%

Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain

Hexnerova

Křížková

Fábry

et al. 2016

Journal of Biological Chemistry

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Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.

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“…Although the degree of selectivity was small, such studies motivated elegant use of non-standard protein engineering to create a novel class of constrained analogs (75). The latter studies employed cell lines that respectively expressed either one isoform or the other but in different lineage-specific contexts.…”

Section: B29mentioning

confidence: 99%

Contribution of TyrB26 to the Function and Stability of Insulin

Pandyarajan¹,

Phillips²,

Rege³

et al. 2016

Journal of Biological Chemistry

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Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of Tyr B26 , a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, nonaromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakestbinding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the Glu B26 analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although Tyr B26 is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences.Insulin, a small globular protein critical to the maintenance of metabolic homeostasis (1), provides a classical model for studies of protein structure and self-assembly (2) with longstanding application to human therapeutics (3). Insulin contains two polypeptide chains, designated A and B, that are linked by two disulfide bridges (cystines A7-B7 and A20-B19); the A chain is further stabilized by an intrachain bridge (cystine A6-A11). In pancreatic ␤-cells, insulin is stored within glucoseregulated secretory granules as zinc-coordinated hexamers (Fig. 1A). The hormone binds to a receptor-tyrosine kinase, designated the insulin receptor (IR). 5 The product of a single gene, the IR precursor is processed in the trans-Golgi network into its final disulfide-linked (␣␤) 2 homodimer conformation. The extracellular ␣ subunit contains hormone-binding elements, whereas the transmembrane ␤ subunit contains the intracellular tyrosine kinase domain (4). Alternative splicing leads to two receptor isoforms, IR-A and IR-B (5). The threedimensional structure of the holoreceptor has been visualized only at low resolution (Ͼ20 Å), but these findings have been inconclusive (for review, see Ref. 6). How extracellular binding of insulin alters the structure of the receptor, leading in turn to activation of the intracellular tyrosine kinase domains, is a major unsolved problem (7).Dissection of the IR into discrete domains has enabled crystallographic analysis of its parts. The relevant structural biology is as follows. (i) Structures of the intracellular tyrosine kinase domains at 1.9 Å resolution have been o...

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Rational steering of insulin binding specificity by intra-chain chemical crosslinking

Cited by 20 publications

References 56 publications

Structural Perspectives of Insulin Receptor Isoform-Selective Insulin Analogs

Structural Perspectives of Insulin Receptor Isoform-Selective Insulin Analogs

Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain

Contribution of TyrB26 to the Function and Stability of Insulin

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