Non-equivalent role of inter- and intramolecular hydrogen bonds in the insulin dimer interface

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. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 μM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 μM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.     

Modulation of the antagonistic properties of an insulin mimetic peptide by disulfide bridge modifications

Insulin is a peptide responsible for regulating the metabolic homeostasis of the organism; it elicits its effects through binding to the transmembrane insulin receptor (IR). Insulin mimetics with agonistic or antagonistic effects toward the receptor are an exciting field of research and could find applications in treating diabetes or malignant diseases. We prepared five variants of a previously reported 20-amino acid insulin-mimicking peptide. These peptides differ from each other by the structure of the covalent bridge connecting positions 11 and 18. In addition to the peptide with a disulfide bridge, a derivative with a dicarba bridge and three derivatives with a 1,2,3-triazole differing from each other by the presence of sulfur or oxygen in their staples were prepared. The strongest binding to IR was exhibited by the peptide with a disulfide bridge. All other derivatives only weakly bound to IR, and a relationship between increasing bridge length and lower binding affinity can be inferred. Despite their nanomolar affinities, none of the prepared peptide mimetics was able to activate the insulin receptor even at high concentrations, but all mimetics were able to inhibit insulin-induced receptor activation. However, the receptor remained approximately 30% active even at the highest concentration of the agents; thus, the agents behave as partial antagonists. An interesting observation is that these mimetic peptides do not antagonize insulin action in proportion to their binding affinities. The compounds characterized in this study show that it is possible to modulate the functional properties of insulin receptor peptide ligands using disulfide mimetics.     

Mechanism of transmembrane signaling: insulin binding and the insulin receptor

Transmembrane signaling via receptor tyrosine kinases generally requires oligomerization of receptor monomers, with the formation of ligand-induced dimers or higher multimers of the extracellular domains of the receptors. Such formations are expected to juxtapose the intracellular kinase domains at the correct distances and orientations for transphosphorylation. For receptors of the insulin receptor family that are constitutively dimeric, or those that form noncovalent dimers without ligands, the mechanism must be more complex. For these, the conformation must be changed by the ligand from one that prevents activation to one that is permissive for kinase phosphorylation. How the insulin ligand accomplishes this action has remained a puzzle since the discovery of the insulin receptor over 2 decades ago, primarily because membrane proteins in general have been refractory to structure determination by crystallography. However, high-resolution structural evidence on individual separate subdomains of the insulin receptor and of analogous proteins has been obtained. The recently solved quaternary structure of the complete dimeric insulin receptor in the presence of insulin has now served as the structural envelope into which such individual domains were fitted. The combined structure has provided answers on the details of insulin/receptor interactions in the binding site and on the mechanism of transmembrane signaling of this covalent dimer. The structure explains many observations on the behavior of the receptor, from greater or lesser binding of insulin and its variants, point and deletion mutants of the receptor, to antibody-binding patterns, and to the effects on basal and insulin-stimulated autophosphorylation under mild reducing conditions.     

X-ray structure of human relaxin at 1.5 A. Comparison to insulin and implications for receptor binding determinants.

The X-ray crystal structure of relaxin at 1.5 A resolution is reported for the physiologically active form of the human hormone. Relaxin is a small, two-chain polypeptide that is a member of the protein hormone family that also includes insulin and the insulin-like growth factors IGF-I and IGF-II. These hormones have biologically diverse activities but are structurally similar, sharing a distinctive pattern of cysteine and glycine residues. The predicted structural homology of relaxin to insulin is confirmed by this structural analysis; however, there are significant differences in the terminal regions of the b-chain. Although relaxin, like insulin, crystallizes as a dimer, the orientation of the molecules in the respective dimers is completely different. The region of the relaxin molecule proposed to be involved in receptor binding is part of the dimer interface, suggesting that some of the other residues contained in the dimer contact surface might be receptor binding determinants as well. The proposed receptor binding determinants for insulin likewise include residues at its dimer interface. However, because the dimer contacts of relaxin and insulin are quite different, it appears that these two structurally related hormones have evolved somewhat dissimilar mechanisms for receptor binding.     

Molecular complementarity III. peptide complementarity as a basis for peptide receptor evolution: a bioinformatic case study of insulin,glucagon and gastrin

Dwyer has suggested that peptide receptors evolved from self-aggregating peptides so that peptide receptors should incorporate regions of high homology with the peptide ligand. If one considers self-aggregation to be a particular manifestation of molecular complementarity in general, then it is possible to extend Dwyer's hypothesis to a broader set of peptides: complementary peptides that bind to each other. In the latter case, one would expect to find homologous copies of the complementary peptide in the receptor. Thirteen peptides, 10 of which are not known to self-aggregate (amylin, ACTH, LHRH, angiotensin II, atrial natriuretic peptide, somatostatin, oxytocin, neurotensin, vasopressin, and substance P), and three that are known to self-aggregate (insulin, glucagon, and gastrin), were chosen. In addition to being self-aggregating, insulin and glucagon are also known to bind to each other, making them a mutually complementary pair. All possible combinations of the 13 peptides and the extracellular regions of their receptors were investigated using bioinformatic tools (a total of 325 combinations). Multiple, statistically significant homologies were found for insulin in the insulin receptor; insulin in the glucagon receptor; glucagon in the glucagon receptor; glucagon in the insulin receptor; and gastrin in gastrin binding protein and its receptor. Most of these homologies are in regions or sequences known to contribute to receptor binding of the respective hormone. These results suggest that the Dwyer hypothesis for receptor evolution may be generalizable beyond self-aggregating to complementary peptides. The evolution of receptors may have been driven by small molecule complementarity augmented by modular evolutionary processes that left a "molecular paleontology" that is still evident in the genome today. This "paleontology" may allow identification of peptide receptor sites.     

The cysteine-rich domains of the insulin and insulin-like growth factor I receptors are primary determinants of hormone binding specificity. Evidence from receptor chimeras

To delineate the structural determinants of the insulin receptor (IR) and insulin-like growth factor I receptor (IGFIR) which affect hormone binding specificity we have constructed seven chimeric receptor cDNAs and stably expressed them in Chinese hamster ovary cells. Clonal cell lines expressing high levels of each receptor chimera were analyzed for insulin and insulin-like growth factor I (IGFI) binding activity. Measurements of hormone binding and immunoprecipitation of metabolically labeled receptors showed that all chimeras were properly processed and expressed at the cell surface. The binding data indicate that 56 amino acids of the IR and 52 amino acids of the IGFIR located in corresponding regions of the cysteine-rich domains are the primary determinants of hormone binding specificity. These regions are located between amino acids Asn-230 and Ile-285 on the IR and between His-223 and Met-274 on the IGFIR. In addition, the alpha IR-3 antibody, which competes for IGFI binding, was found to interact with the same 52 amino acids of the IGFIR which determines hormone specificity. Other antibodies which interfere with insulin binding (5D9, MC51, and MA20) interact with epitopes in the COOH-terminal 288 amino acids of the alpha-subunit. We conclude that 56 and 52 amino acids of the cysteine-rich domains of the IR and IGFIR contain the major determinants of hormone binding specificity although other more COOH-terminal regions of both receptors contribute to hormone binding.     

Binding and autophosphorylating activity of human insulin analogs

Insulin receptor binding and autophosphorylating activities of a number of synthetic analogs of human insulin have been examined using highly purified insulin receptor from human placenta. In general, autophosphorylation correlates well with the ability of the analogs to stimulate glucose oxidation and to inhibit lipolysis in adipocytes although their biological activities varied over a wide range. These findings support the hypothesis that autophosphorylation is an obligatory step in the pathways leading to glucose oxidation and inhibition of lipolysis. The relative biological potencies of the analogs in the autophosphorylation assay also correlated well with their receptor-binding affinities except for the peptides [endo-TyrB16a]insulin, in which an additional Tyr has been inserted between TyrB16 and LeuB17 and [ProA2]insulin. The relative receptor binding affinity of [endo-TyrB16a]insulin is significantly greater than its biological activity in the adipocyte or receptor autophosphorylation assays. The converse is true for [ProA2]insulin. These results demonstrate that the amino-acid residues involved in binding and receptor activation may not be identical.     

Structural requirements for the transmembrane activation of the insulin receptor kinase

Tetrameric insulin holoreceptor (alpha 2 beta 2) was reduced with dithiothreitol into alpha beta dimers such that they maintain up to 50% of insulin binding at tracer ligand concentrations. Scatchard analysis of insulin binding to dimers revealed that they had a reduced affinity for ligand by a factor of 3-6 compared to holoreceptor, whereas the maximum number of high affinity binding sites was not affected. The alpha beta dimers can be separated from holoreceptor by sucrose density gradient centrifugation, and hence, they are not associated by noncovalent interactions. Insulin-dependent autophosphorylation of alpha beta dimers isolated from low ionic strength sucrose density gradients was minimal and was always accompanied by reoxidation of dimers to the tetrameric holoreceptor. The reformed tetramer exhibited a strong insulin-dependent autophosphorylation reaction. Reoxidation was prevented by isolating alpha beta dimers in sucrose density gradients containing 0.15 M NaCl. Under these conditions, no insulin-dependent autophosphorylation was observed. When insulin receptor was first autophosphorylated and then reduced, receptor kinase activity, as assayed by histone phosphorylation, was not affected. Also, the insulin-independent, basal autophosphorylation was maintained after reduction into alpha beta dimers. We conclude that alpha beta-alpha beta interaction is not necessary for the maintenance of basal kinase activity or for insulin-activated kinase activity once autophosphorylation occurs. However, dimer-dimer interaction appears critical for the insulin-dependent activation of the receptor's intrinsic kinase activity.     

Cis- and trans-activation of hormone receptors: the LH receptor

G protein-coupled receptors (GPCRs) accommodate a wide spectrum of activators from ions to glycoprotein hormones. The mechanism of activation for this large and clinically important family of receptors is poorly understood. Although initially thought to function as monomers, there is a growing body of evidence that GPCR dimers form, and in some cases that these dimers are essential for signal transduction. Here we describe a novel mechanism of intermolecular GPCR activation, which we refer to as trans-activation, in the LH receptor, a GPCR that does not form stable dimers. The LH receptor consists of a 350-amino acid amino-terminal domain, which is responsible for high-affinity binding to human CG, followed by seven-transmembrane domains and connecting loops. This seven-transmembrane domain bundle transmits the signal from the extracellular amino terminus to intracellular G proteins and adenylyl cyclase. Here, we show that binding of hormone to one receptor can activate adenylyl cyclase through its transmembrane bundle, intramolecular activation (cis-activation), as well as trans-activation through the transmembrane bundle of an adjacent receptor, without forming a stable receptor dimer. Coexpression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescues hormone-activated cAMP production. Our observations provide new insights into the mechanism of receptor activation mechanisms and have implications for the treatment of inherited disorders of glycoprotein hormone receptors.     

Peptides identify the critical hotspots involved in the biological activation of the insulin receptor

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.     

Diversity in peptide recognition by the SH2 domain of SH2B1

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein‐protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate‐1 and ?2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C‐terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high‐resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand‐binding interface of SH2B1 with high specificity.     

Polylysine increases the number of insulin binding sites in soluble insulin receptor preparations

The effects of cationic polyamino acids on insulin binding to soluble insulin receptor preparations were studied. Incubation of partially or fully purified receptor preparations with polylysine (pLys) increased by several-fold the amount of [125I]insulin that remained associated with the receptor, as determined both by precipitation of receptor-insulin complexes by polyethylene glycol or by separation of the complexes from the free hormone by gel filtration. This elevation in the amount of bound insulin resulted from increased number of insulin binding sites, and could not be attributed to an increased affinity of the receptors to insulin. In fact, pLys reduced 2-3-fold the affinity of insulin binding to its receptor as determined by equilibrium binding studies, and by monitoring the rate of exchange of bound [125I]insulin with unlabeled hormone. pLys induced specific interactions between insulin and its native receptor since other basic compounds such as histone, spermidine, polymixin B, compound 48/80, lysine, and arginine failed to reproduce its effects. pLys did not interact with the free ligand, nor did it promote interactions between insulin and denatured receptor forms. Furthermore, pLys did not induce binding of insulin to other proteins present in the partially purified receptor preparations. The effects of pLys were time and dose-dependent and were proportional to the pLys chain length. The longer the chain, the greater was the effect. Enhanced insulin binding and receptor beta-subunit autophosphorylation (in the presence of insulin) exhibited a similar dependency on the chain length of pLys. pLys effects on insulin binding were associated with formation of large protein aggregates that remained trapped at the top of Sephacryl S-300 columns. These aggregates contained substantial amounts of receptor-insulin complexes. Our results suggest that pLys induces formation of receptor clusters that create de novo insulin binding sites among adjacent receptor tetramers. Alternatively, formation of receptor aggregates might facilitate insulin binding to a soluble receptor subfraction that otherwise fails to bind the hormone.     

The binding ability of insulin-related peptides as the clue to the search for their functional role in phylogenesis. Introduction to the problem

The common plan of structure of the main peptides of the vertebrate insulin family—insulin itself, IGF-I, IGF-II, and relaxin—has distinct structural formations. Each of the peptides performs its characteristic function. However, overlapping of insulin and IGF-I actions and its stability in the vertebrate phylogenesis have formed the concept of their regulation of growth and metabolism as a function fixed in phylogenesis for a certain type of structures. At the same time, study of insulin-related peptides in invertebrates has revealed the wider spectrum, than in vertebrates, of biological effects; this indicated that the similarity of the total structure design is not sufficient for judging about their functional role. Functional possibilities of a regulatory peptide depend fundamentally on its capability for binding to the receptor realizing its biological action. However, the binding ability has a wider significance than merely transmission of biological signals. Thus, IGF-II when interacting with receptors realizing its biological effects also binds to the IGF-2 receptor limiting its action and, besides, to the binding proteins (BP) modulating its action. The entire cycle of interactions occurs in the body at different affinity levels. Meanwhile, insulin interacts neither with IGF-2 receptor nor with BP. In this case, specificity and sequence of interaction with each of receptors or with protein are due not to the general design of the peptide structure, but rather to structure of individual submolecular determinants—binding domains. The leading role in disclosure of composition and structure of these domains is played by the “mutant-ligand” approach evaluating affinity of modified analogs. To analyze role of structural elements of the binding domains, the author proposes the system of estimation of affinity of the studied analogs. The present work, alongside with consideration of methodical aspects of the forthcoming analysis, is an introduction to the problem of organization of the binding domains connected directly with functional role of peptides of the insulin type. The proposed analysis is due to necessity of specification of this organization both in one molecule and in different molecules with a similar plan of structure on the basis of not always unanimous literature data and of clarification of principles of structure of these domains.     

The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer

Extracts containing wild-type or mutant human estrogen receptor (ER) have been used to study the binding of ER to its responsive element (ERE). Estradiol (E2) or the antiestrogen hydroxytamoxifen is required for ER binding as assayed by gel retardation. The DNA binding domain (DBD) encompasses the highly conserved region C. Both intact ER-E2 complexes and ER mutants truncated for the hormone binding domain (HBD) bind as dimers to an ERE. However, an HBD-truncated ER binds less tightly to an ERE than an intact ER-E2 complex. The DBD and HBD contain a constitutive and a stronger ER-induced dimerization function, respectively. Thus, in addition to inducing the activation function associated with the HBD, estrogen plays a crucial role in the formation of stable ER dimers that bind tightly to ERE.     

Inhibition of autophosphorylation of epidermal growth factor receptor by a small peptide not employing an ATP-competitive mechanism

Previously we found that short peptides surrounding major autophosphorylation sites of EGFR (VPEY(1068)INQ, DY(1148)QQD, and ENAEY(1173)LR) suppress phosphorylation of purified EGFR to 30-50% at 4000 microM. In an attempt to improve potencies of the peptides, we modified the sequences by substituting various amino acids for tyrosine or by substituting Gln and Asn for Glu and Asp, respectively. Among the modified peptides, Asp/Asn- and Glu/Gln-substitution in DYQQD (NYQQN) and ENAEYLR (QNAQYLR), respectively, improved inhibitory potencies. The inhibitory potency of NYQQN was not affected by the concentration of ATP, while that of QNAQYLR was affected. Docking simulations showed different mechanisms of inhibition for the peptides: inhibition by binding to the ATP-binding site (QNAQYLR) and inhibition by binding to a region surrounded by alphaC, the activation loop, and the catalytic loop and interfering with the catalytic reaction (NYQQN). The inhibitory potency of NYQQN for insulin receptor drastically decreased, whereas QNAQYLR inhibited autophosphorylation of insulin receptor as well as EGFR. In conclusion, NYQQN is not an ATP-competitive inhibitor and the binding site of this peptide appears to be novel as a tyrosine kinase inhibitor. NYQQN could be a promising seed for the development of anti-cancer drugs having specificity for EGFR.     

Binding of insulin-like growth Factor ii and multiplication-stimulating activity-stimulated phosphorylation in basolateral membranes from dog kidney

Biologic actions of insulin and insulin-like growth factors (IGFs) are thought to be initiated by binding of peptides to tissues, followed by phosphorylation of specific hormone receptors. Both insulin and IGF bind to renal membranes, suggesting functional roles for these peptides in kidney. The present studies further characterize the interaction of multiplication-stimulating activity (MSA)/IGF II with its renal receptor. Specific binding of 125I-IGF II was measured in basolateral membranes isolated from proximal tubular cells of dog kidney. Binding was half-maximal at 10(-9) M MSA and was not inhibited by human growth hormone, IGF I, insulin, or anti-insulin receptor antibodies. Concentration-dependent MSA-stimulated phosphorylation of a Mr 135,000 protein band was demonstrated in autoradiograms of sodium dodecyl sulfate-polyacrylamide gels from basolateral membrane suspensions. Insulin increased phosphorylation of this band only in the presence of MSA, while a Mr 92,000 band was consistently phosphorylated with insulin alone. The phosphorylated Mr 135,000 band which had been solubilized with detergent from basolateral membranes was immunoprecipitated using serum from a patient with anti-insulin receptor antibodies suggesting that the band is the alpha subunit of the insulin receptor. This was supported by the demonstration of covalent cross-linkage of 125I-insulin to the Mr 135,000 band. We conclude that receptor-mediated MSA-stimulated phosphorylation of isolated basolateral membranes may reflect a process by which biological actions of IGF II are mediated in vivo. Our data suggest that insulin and IGF II may interact by regulating protein phosphorylation.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its beta-subunit: possible involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding of the hormone.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Construction and characterization of a monomeric insulin receptor

A mutant insulin receptor was constructed by replacing cysteine residues Cys(524), Cys(682), Cys(683), and Cys(685) with serine. The mutant was expressed in COS7 and Chinese hamster ovary cells, did not form covalently linked dimers, and was present at the cell surface. There was half as much insulin binding activity at the cell surface in cells expressing the mutant compared with that in cells expressing the wild type receptor. The intracellular processing of the mutant receptor was affected, since its beta-subunit migrated more slowly than that of the wild type receptor on SDS-PAGE. The mutant was capable of insulin-dependent autophosphorylation and phosphorylation of insulin receptor substrate-1 in vivo and could be cross-linked into receptor dimers when membrane-bound. The amount of insulin-dependent autophosphorylation of the mutant receptor was half that of the wild type receptor. However, after solubilization the monomeric insulin receptor had minimal autophosphorylation activity, and, unlike the naturally occurring monomeric receptor tyrosine kinases, the solubilized monomeric insulin receptor did not dimerize in response to insulin binding as determined by sucrose density gradient centrifugation.