ABSTRACT
Insulin is a mainstay of therapy for diabetes mellitus, yet its thermal stability complicates global transportation and storage. Cold-chain transport, coupled with optimized formulation and materials, prevents to some degree nucleation of amyloid and hence inactivation of hormonal activity. These issues hence motivate the design of analogs with increased stability, with a promising approach being single-chain insulins (SCIs), whose C domains (foreshortened relative to proinsulin) resemble those of the single-chain growth factors (IGFs). We have previously demonstrated that optimized SCIs can exhibit native-like hormonal activity with enhanced thermal stability and marked resistance to fibrillation. Here, we describe the crystal structure of an ultrastable SCI (C-domain length 6; sequence EEGPRR) bound to modules of the insulin receptor (IR) ectodomain (N-terminal α-subunit domains L1-CR and C-terminal αCT peptide; "microreceptor" [µIR]). The structure of the SCI-µIR complex, stabilized by an Fv module, was determined using diffraction data to a resolution of 2.6 Å. Remarkably, the αCT peptide (IR-A isoform) "threads" through a gap between the flexible C domain and the insulin core. To explore such threading, we undertook molecular dynamics simulations to 1) compare threaded with unthreaded binding modes and 2) evaluate effects of C-domain length on these alternate modes. The simulations (employing both conventional and enhanced sampling simulations) provide evidence that very short linkers (C-domain length of -1) would limit gap opening in the SCI and so impair threading. We envisage that analogous threading occurs in the intact SCI-IR complex-rationalizing why minimal C-domain lengths block complete activity-and might be exploited to design novel receptor-isoform-specific analogs.
Subject(s)
Insulin , Receptor, Insulin , Receptor, Insulin/metabolism , Insulin/metabolism , Models, Molecular , Protein Binding , Peptides/chemistryABSTRACT
Monomers of the insulin receptor and type 1 insulin-like growth factor receptor (IGF-1R) can combine stochastically to form heterodimeric hybrid receptors. These hybrid receptors display ligand binding and signaling properties that differ from those of the homodimeric receptors. Here, we describe the cryoelectron microscopy structure of such a hybrid receptor in complex with insulin-like growth factor I (IGF-I). The structure (ca. 3.7 Å resolution) displays a single IGF-I ligand, bound in a similar fashion to that seen for IGFs in complex with IGF-1R. The IGF-I ligand engages the first leucine-rich-repeat domain and cysteine-rich region of the IGF-1R monomer (rather than those of the insulin receptor monomer), consistent with the determinants for IGF binding residing in the IGF-1R cysteine-rich region. The structure broadens our understanding of this receptor family and assists in delineating the key structural motifs involved in binding their respective ligands.
Subject(s)
Insulin-Like Growth Factor I , Receptor, Insulin , Cryoelectron Microscopy , Cysteine , Insulin/metabolism , Insulin-Like Growth Factor I/metabolism , Ligands , Receptor, IGF Type 1/chemistry , Receptor, Insulin/chemistry , Receptor, Insulin/metabolism , Receptors, SomatomedinABSTRACT
Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.
Subject(s)
Insulin , Mollusk Venoms , Cryoelectron Microscopy , Humans , Insulin/metabolism , Mollusk Venoms/chemistry , Mollusk Venoms/metabolism , Peptides , Protein ConformationABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.
Subject(s)
Antigens, CD/chemistry , Insulin/chemistry , Mollusk Venoms/chemistry , Mollusk Venoms/metabolism , Receptor, Insulin/chemistry , Amino Acid Substitution , Animals , Antigens, CD/metabolism , Binding Sites , Crystallography, X-Ray , Insulin/analogs & derivatives , Insulin/metabolism , Insulin/pharmacology , Mice, Inbred C57BL , Models, Molecular , Molecular Dynamics Simulation , Mollusk Venoms/genetics , Mollusk Venoms/pharmacology , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Conformation , Rats, Sprague-Dawley , Receptor, Insulin/metabolism , Structure-Activity Relationship , TyrosineABSTRACT
Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 Å in the insulin-binding region and 4.2 Å in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin's negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design.
Subject(s)
Receptor, Insulin/chemistry , Receptor, Insulin/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Protein Binding , Protein Conformation , Protein Structure, Secondary , Receptor, Insulin/metabolism , Signal Transduction/physiologyABSTRACT
Human type 1 insulin-like growth factor receptor is a homodimeric receptor tyrosine kinase that signals into pathways directing normal cellular growth, differentiation and proliferation, with aberrant signalling implicated in cancer. Insulin-like growth factor binding is understood to relax conformational restraints within the homodimer, initiating transphosphorylation of the tyrosine kinase domains. However, no three-dimensional structures exist for the receptor ectodomain to inform atomic-level understanding of these events. Here, we present crystal structures of the ectodomain in apo form and in complex with insulin-like growth factor I, the latter obtained by crystal soaking. These structures not only provide a wealth of detail of the growth factor interaction with the receptor's primary ligand-binding site but also indicate that ligand binding separates receptor domains by a mechanism of induced fit. Our findings are of importance to the design of agents targeting IGF-1R and its partner protein, the human insulin receptor.
Subject(s)
Insulin-Like Growth Factor I/chemistry , Receptors, Somatomedin/chemistry , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Cricetulus , Crystallography, X-Ray , Gene Expression , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Kinetics , Ligands , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptor, IGF Type 1 , Receptors, Somatomedin/genetics , Receptors, Somatomedin/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sf9 Cells , SpodopteraABSTRACT
The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin's ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues.
Subject(s)
Disulfides/chemistry , Insulin/chemistry , Insulin/metabolism , Allosteric Regulation , Amino Acid Sequence , Humans , Molecular Dynamics Simulation , Protein Conformation , StereoisomerismABSTRACT
Insulin, a protein critical for metabolic homeostasis, provides a classical model for protein design with application to human health. Recent efforts to improve its pharmaceutical formulation demonstrated that iodination of a conserved tyrosine (TyrB26) enhances key properties of a rapid-acting clinical analog. Moreover, the broad utility of halogens in medicinal chemistry has motivated the use of hybrid quantum- and molecular-mechanical methods to study proteins. Here, we (i) undertook quantitative atomistic simulations of 3-[iodo-TyrB26]insulin to predict its structural features, and (ii) tested these predictions by X-ray crystallography. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog, as a dimer and hexamer, exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Aromatic rings (TyrB16, PheB24, PheB25, 3-I-TyrB26, and their symmetry-related mates) at this interface adjust to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of a 3-[iodo-TyrB26]insulin analog (determined as an R6 zinc hexamer). Given that residues B24-B30 detach from the core on receptor binding, the environment of 3-I-TyrB26 in a receptor complex must differ from that in the free hormone. Based on the recent structure of a "micro-receptor" complex, we predict that 3-I-TyrB26 engages the receptor via directional halogen bonding and halogen-directed hydrogen bonding as follows: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient σ-hole and electronegative equatorial band. Inspired by quantum chemistry and molecular dynamics, such "halogen engineering" promises to extend principles of medicinal chemistry to proteins.
Subject(s)
Chemistry, Pharmaceutical , Insulin/analogs & derivatives , Receptor, Insulin/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Halogens , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Insulin/chemistry , Insulin/genetics , Insulin/metabolism , Models, Molecular , Phenylalanine/chemistry , Phenylalanine/genetics , Phenylalanine/metabolism , Protein Binding , Receptor, Insulin/chemistry , Structure-Activity Relationship , Tyrosine/chemistry , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
Insulins in the venom of certain fish-hunting cone snails facilitate prey capture by rapidly inducing hypoglycemic shock. One such insulin, Conus geographus G1 (Con-Ins G1), is the smallest known insulin found in nature and lacks the C-terminal segment of the B chain that, in human insulin, mediates engagement of the insulin receptor and assembly of the hormone's hexameric storage form. Removal of this segment (residues B23-B30) in human insulin results in substantial loss of receptor affinity. Here, we found that Con-Ins G1 is monomeric, strongly binds the human insulin receptor and activates receptor signaling. Con-Ins G1 thus is a naturally occurring B-chain-minimized mimetic of human insulin. Our crystal structure of Con-Ins G1 reveals a tertiary structure highly similar to that of human insulin and indicates how Con-Ins G1's lack of an equivalent to the key receptor-engaging residue PheB24 is mitigated. These findings may facilitate efforts to design ultrarapid-acting therapeutic insulins.
Subject(s)
Antigens, CD/metabolism , Conus Snail/metabolism , Insulin/metabolism , Receptor, Insulin/metabolism , Venoms/metabolism , Amino Acid Sequence , Animals , Antigens, CD/chemistry , Humans , Insulin/chemistry , Models, Molecular , Protein Binding , Protein Conformation , Protein Multimerization , Receptor, Insulin/chemistry , Selenocysteine/chemistry , Selenocysteine/metabolism , Venoms/chemistryABSTRACT
Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe(701) and Phe(705) The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor.
Subject(s)
Biomimetic Materials/chemistry , Insulin/chemistry , Peptide Library , Receptor, Insulin/chemistry , Amino Acid Motifs , Animals , Binding Sites , Humans , MiceABSTRACT
The homodimeric insulin and type 1 insulin-like growth factor receptors (IR and IGF-1R) share a common architecture and each can bind all three ligands within the family: insulin and insulin-like growth factors I and II (IGF-I and IFG-II). The receptor monomers also assemble as heterodimers, the primary ligand-binding sites of which each comprise the first leucine-rich repeat domain (L1) of one receptor type and an α-chain C-terminal segment (αCT) of the second receptor type. We present here crystal structures of IGF-I bound to such a hybrid primary binding site and of a ligand-free version of an IR αCT peptide bound to an IR L1 plus cysteine-rich domain construct (IR310.T). These structures, refined at 3.0-Å resolution, prove congruent to respective existing structures of insulin-complexed IR310.T and the intact apo-IR ectodomain. As such, they provide key missing links in the emerging, but sparse, repertoire of structures defining the receptor family.
Subject(s)
Insulin-Like Growth Factor I/chemistry , Receptor, IGF Type 1/chemistry , Receptor, Insulin/chemistry , Binding Sites , Crystallography, X-Ray , Humans , Ligands , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, SecondaryABSTRACT
Crystallographic studies of insulin bound to fragments of the insulin receptor have recently defined the topography of the primary hormone-receptor interface. Here, we have investigated the role of Phe(B24), an invariant aromatic anchor at this interface and site of a human mutation causing diabetes mellitus. An extensive set of B24 substitutions has been constructed and tested for effects on receptor binding. Although aromaticity has long been considered a key requirement at this position, Met(B24) was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchor at the hormone-receptor interface. These findings motivated further substitution of Phe(B24) by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [Cha(B24)]insulin likewise exhibited high activity. Furthermore, its resistance to fibrillation and the rapid rate of hexamer disassembly, properties of potential therapeutic advantage, were enhanced. The crystal structure of the Cha(B24) analog, determined as an R6 zinc-stabilized hexamer at a resolution of 1.5 Å, closely resembles that of wild-type insulin. The nonplanar aliphatic ring exhibits two chair conformations with partial occupancies, each recapitulating the role of Phe(B24) at the dimer interface. Together, these studies have defined structural requirements of an anchor residue within the B24-binding pocket of the insulin receptor; similar molecular principles are likely to pertain to insulin-related growth factors. Our results highlight in particular the utility of nonaromatic side chains as probes of the B24 pocket and suggest that the nonstandard Cha side chain may have therapeutic utility.
Subject(s)
Drug Design , Insulin/chemistry , Insulin/metabolism , Phenylalanine , Receptor, Insulin/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , Humans , Hydrophobic and Hydrophilic Interactions , Insulin/genetics , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Folding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Receptor, IGF Type 1/chemistry , Receptor, IGF Type 1/metabolism , Receptor, Insulin/chemistryABSTRACT
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 B-chain segment, critical for protective self-assembly in ß 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 ß-turn, coupling reorientation of Phe(B24) to a 60° rotation of the B25-B28 ß-strand away from the hormone core to lie antiparallel to the receptor's L1-ß2 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 receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.
Subject(s)
Insulin/metabolism , Receptor, Insulin/metabolism , Crystallography, X-Ray , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein BindingABSTRACT
Unraveling the molecular detail of insulin receptor activation has proved challenging, but a major advance is the recent determination of crystallographic structures of insulin in complex with its primary binding site on the receptor. The current model for insulin receptor activation is that two distinct surfaces of insulin monomer engage sequentially with two distinct binding sites on the extracellular surface of the insulin receptor, which is itself a disulfide-linked (αß)2 homodimer. In the process, conformational changes occur both within the hormone and the receptor, the latter resulting in the disruption of the intracellular interactions that hold the kinase domains in their basal state and in the initiation of the phosphorylation events that drive insulin signaling. The purpose of this paper is to summarize the extant structural data relating to hormone binding and how it effects receptor activation, as well as to discuss the issues that remain unresolved.
Subject(s)
Protein Binding/physiology , Receptor, IGF Type 1/metabolism , Receptor, Insulin/metabolism , Binding Sites , Humans , Insulin/metabolism , Models, Molecular , Phosphorylation , Protein Structure, Tertiary , Protein-Tyrosine Kinases/metabolism , Sequence Analysis, DNA , Signal TransductionABSTRACT
Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer's disease; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin-insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal ß-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone-receptor recognition is novel within the broader family of receptor tyrosine kinases. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone-insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.
Subject(s)
Insulin/chemistry , Insulin/metabolism , Receptor, Insulin/chemistry , Receptor, Insulin/metabolism , Animals , Binding Sites , Calorimetry , Cattle , Cell Line , Crystallography, X-Ray , Humans , Leucine/metabolism , Ligands , Models, Molecular , Protein Binding , Protein Structure, Secondary , Reproducibility of ResultsABSTRACT
The C-terminal segment of the human insulin receptor alpha-chain (designated alphaCT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-cross-linking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The alphaCT segment is now resolved as being engaged with the central beta-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is alpha-helical in conformation and extends 11 residues N-terminal of the classical alphaCT segment boundary originally defined by peptide mapping. This tandem structural element (alphaCT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant alphaCT peptides binding to an insulin minireceptor, leads to the conclusion that putative "insulin-mimetic" peptides in the literature act at least in part as mimics of the alphaCT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the alphaCT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate alpha-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus.
Subject(s)
Peptides/chemistry , Receptor, Insulin/metabolism , Animals , Binding Sites , CHO Cells , Calorimetry/methods , Cricetinae , Cricetulus , Cross-Linking Reagents/chemistry , Crystallography, X-Ray/methods , Dimerization , Drug Design , Humans , Models, Molecular , Molecular Conformation , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Receptor, Insulin/agonistsABSTRACT
The insulin receptor (IR) and the homologous Type 1 insulin-like growth factor receptor (IGF-1R) are cell-surface tyrosine kinase receptors that effect signaling within the respective pathways of glucose metabolism and normal human growth. While ligand binding to these receptors is assumed to result in a structural transition within the receptor ectodomain that then effects signal transduction across the cell membrane, little is known about the molecular detail of these events. Presented here are small-angle X-ray scattering data obtained from the IR and IGF-1R ectodomains in solution. We show that, in solution, the ectodomains of IR and IGF-1R have a domain disposition that is very similar to that seen in the crystal structure of the ectodomain of IR, despite the constituent domains being in relatively sparse contact and potentially mobile. We also show that the IGF-1R ectodomain is capable of binding up to three molecules of IGF-1 in solution, with surprisingly little apparent change in relative domain disposition compared to the apo form. While the observed 3:1 ligand-binding stoichiometry appears to contradict earlier explanations of the absence of a bell-shaped dose-response curve for IGF-1R in ligand displacement assays, it is readily understood in the context of the harmonic oscillator model of the negative cooperativity of ligand binding to IGF-1R. Taken together, our findings suggest that the structural movements within these receptors upon ligand binding are small and are possibly limited to local rotation of domains.
Subject(s)
Antigens, CD/chemistry , Insulin-Like Growth Factor I/metabolism , Insulin/metabolism , Receptor, IGF Type 1/chemistry , Receptor, Insulin/chemistry , Animals , Antigens, CD/metabolism , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, Quaternary , Protein Structure, Tertiary , Receptor, IGF Type 1/metabolism , Receptor, Insulin/metabolism , Scattering, Small AngleABSTRACT
The C-terminal segment of the insulin receptor (IR) alpha-chain plays a critical role in insulin binding. This 16-residue peptide together with the central beta-sheet of the receptor L1 domain forms one of the insulin binding surfaces of the IR monomer. Here we use isothermal titration calorimetry to assay directly the binding of the IR alphaCT peptide to an IR construct (IR485) consisting of the three N-terminal domains of the receptor monomer. Our measurements show further that the binding of the IR alphaCT peptide to IR485 competes with the binding of a prototypical "Site 1" insulin mimetic peptide to the same receptor fragment. The competitive nature of their binding appears to be reflected in a previously undetected sequence similarity between the IR alphaCT peptide and the Site 1 mimetic peptide. In contrast, a prototypical "Site 2" peptide has very limited affinity for IR485. Taken together, these results complement our recent observation that there is a possible structural relationship between these mimetic peptides and insulin itself. They also add support to the view that the segment of unexplained electron density lying on the surface of the central beta-sheet of the L1 domain in the IR ectodomain crystal structure arises from the IR alphaCT peptide. Finally, we show that mutation of the critical IR alphaCT peptide residue Phe714 to alanine does not affect the peptide's affinity for IR485 and conclude that the resultant loss of insulin binding with this mutation results from loss of interaction of the phenylalanine side chain with insulin.
Subject(s)
Antigens, CD/chemistry , Insulin/chemistry , Peptides/chemistry , Receptor, Insulin/chemistry , Thermodynamics , Animals , Antigens, CD/metabolism , Binding Sites , CHO Cells , Cricetinae , Cricetulus , Humans , Insulin/metabolism , Ligands , Peptides/metabolism , Protein Conformation , Protein Structure, Tertiary , Receptor, Insulin/metabolismABSTRACT
Saturation transfer difference (STD) (1)H NMR experiments were used to probe the epitope binding characteristics of the sialidase [EC 3.2.1.18] from the bacterium Vibrio cholerae, the causative agent of cholera. Binding preferences were investigated for N-acetylneuraminic acid (Neu5Ac, 1), the product of the sialidase catalytic reaction, for the known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (Neu5Ac2en, 2), and for the uronic acid-based Neu5Ac2en mimetic iso-propyl 2-acetamido-2,4-dideoxy-alpha-L-threo-hex-4-enopyranosiduronic acid (3), in which the native glycerol side-chain of Neu5Ac2en is replaced with an O-iso-propyl ether. The STD experiments provided evidence, supporting previous studies, that Neu5Ac (1) binds to the sialidase as the alpha-anomer. Docking experiments using DOCK (version 4.0.1) revealed further information regarding the binding characteristics of the enzyme active site in complex with Neu5Ac2en (2) and the Neu5Ac2en mimetic (3), indicating an expected dominant interaction of the acetamide moiety with the protein.