Structure and Stability of the Insulin Dimer Investigated by Molecular Dynamics Simulation

Abstract

Molecular dynamics simulation indicates that the dynamical behaviour of the insulin dimer is asymmetric. Atomic level knowledge of the interaction modes and protein conformation in the solvation state identifies dynamical structures, held by hydrogen bonds that stabilize, mainly in one monomer, the interaction between the chains. Dynamic cross-correlation analysis shows that the two insulin monomers behave asymmetrically and are almost independent. Solvation energy, calculated to evaluate the contribute of each interface residue to the dimer association pattern, well compares with the experimental association state found in protein mutants indicating that this parameter is an important factor to explain the association properties of mutated insulin dimers.     

Structural Investigations of Full-Length Insulin Receptor Dynamics and Signalling

Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.     

Relationship between insulin receptor subunit association and protein kinase activation: insulin-dependent covalent and Mn/MgATP-dependent noncovalent association of alpha beta heterodimeric insulin receptors into an alpha 2 beta 2 heterotetrameric state

The purified human placenta alpha 2 beta 2 heterotetrameric insulin receptor was reduced and dissociated into a functional alpha beta heterodimeric complex by a combination of alkaline pH and dithiothreitol treatment. In the presence of Mn/MgATP, insulin binding to the isolated alpha beta heterodimeric insulin receptor was found to induce the formation of a covalent disulfide-linked alpha 2 beta 2 heterotetrameric complex. In the absence of insulin, a noncovalent association of the alpha beta heterodimeric insulin receptor complex into an alpha 2 beta 2 heterotetrameric state required the continuous presence of both a divalent metal ion (Mn or Mg) and an adenine nucleotide (ATP, ADP, or AMPPCP). Thus, Mn/MgATP binding and not insulin receptor autophosphorylation was responsible for the noncovalent association into the alpha 2 beta 2 heterotetrameric state. However, the divalent metal ions or NaATP separately was ineffective in inducing the noncovalent association between the alpha beta heterodimers. The specific sulfhydryl agent iodoacetamide (IAN) was observed to inhibit the insulin-dependent covalent association of the alpha beta heterodimers without affecting the Mn/MgATP-induced noncovalent association into the alpha 2 beta 2 heterotetrameric state. Insulin treatment of the isolated alpha beta heterodimeric complex in the presence of IAN demonstrated that the Mn/MgATP-induce noncovalent association into the alpha 2 beta 2 heterotetrameric state was sufficient for insulin stimulation of beta-subunit autophosphorylation and exogenous substrate protein kinase activity. These data indicate that although interaction between the individual insulin receptor alpha beta heterodimers is necessary for insulin stimulation of protein kinase activity it does not require covalent disulfide bond formation.     

Ultrafast dynamics-driven biomolecular recognition where fast activities dictate slow events

In general, biological macromolecules require significant dynamical freedom to carry out their different functions, including signal transduction, metabolism, catalysis and gene regulation. Effectors (ligands, DNA and external milieu, etc) are considered to function in a purely dynamical manner by selectively stabilizing a specific dynamical state, thereby regulating biological function. In particular, proteins in presence of these effectors can exist in several dynamical states with distinct binding or enzymatic activity. Here, we have reviewed the efficacy of ultrafast fluorescence spectroscopy to monitor the dynamical flexibility of various proteins in presence of different effectors leading to their biological activity. Recent studies demonstrate the potency of a combined approach involving picosecond-resolved Förster resonance energy transfer, polarisation-gated fluorescence and time-dependent stokes shift for the exploration of ultrafast dynamics in biomolecular recognition of various protein molecules. The allosteric protein–protein recognition following differential protein–DNA interaction is shown to be a consequence of some ultrafast segmental motions at the C-terminal of Gal repressor protein dimer with DNA operator sequences O_E and O_I. Differential ultrafast dynamics at the C-terminal of λ-repressor protein with two different operator DNA sequences for the protein–protein interaction with different strengths is also reviewed. We have also systemically briefed the study on the role of ultrafast dynamics of water molecules on the functionality of enzyme proteins α-chymotrypsin and deoxyribonuclease I. The studies on the essential ultrafast dynamics at the active site of the enzyme α-chymotrypsin by using an anthraniloyl fluorescent extrinsic probe covalently attached to the serine-195 residue for the enzymatic activity at homeothermic condition has also been reviewed. Finally, we have highlighted the evidence that a photoinduced dynamical event dictates the molecular recognition of a photochromic ligand, dihydroindolizine with the serine protease α-chymotrypsin and with a liposome (L-α-phosphatidylcholine).     

Crystal structure of destripeptide (B28-B30) insulin: implications for insulin dissociation

Destripeptide (B28-B30) insulin (DTRI) is an insulin analogue that has much weaker association ability than native insulin but keeps most of its biological activity. It can be crystallized from a solution containing zinc ions at near-neutral pH. Its crystal structure has been determined by molecular replacement and refined at 1.9 A resolution. DTRI in the crystal exists as a loose hexamer compared with 2Zn insulin. The hexamer only contains one zinc ion that coordinates to the B10 His residues of three monomers. Although residues B28-B30 are located in the monomer-monomer interface within a dimer, the removal of them can simultaneously weaken both the interactions between monomers within the dimer and the interactions between dimers. Because the B-chain C-terminus of insulin is very flexible, we take the DTRI hexamer as a transition state in the native insulin dissociation process and suggest a possible dissociation process of the insulin hexamer based on the DTRI structure.     

The state of association of band 3 protein of the human erythrocyte membrane in solutions of nonionic detergents

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was solubilized and purified in aqueous solutions of two nonionic detergents: Ammonyx-LO (dimethyl laurylamine oxide) and C12E9 (nonaethylene glycol lauryl ether). The state of association of the purified protein was studied by analytical ultracentrifugation. Band 3 protein solubilized and studied in solutions of Ammonyx-LO was found to be in a monomer/dimer/tetramer association equilibrium. Band 3 protein freshly prepared in C12 E9 showed the same behaviour; however, during aging the protein was converted into stable noncovalent dimers. The conversion was retarded by the presence of beta-mercaptoethanol or by treatment of the samples with iodoacetamide; it seems to be due to oxidation of the protein by degradation products of the detergent. It is concluded that a monomer/dimer/tetramer association equilibrium is the native state of association of band 3 protein solubilized by nonionic detergents. Since nonionic detergents are assumed not to interfere with protein-protein interactions among membrane proteins, the results strongly support the claim that, in the erythrocyte membrane, band 3 is in a monomer/dimer/tetramer association equilibrium (Dorst, H.-J. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1605-1618).     

Analysis of the self-association of human red cell spectrin

The self-association equilibrium of spectrin has been studied by separating the molecular species present in the cooled reaction mixture by gel electrophoresis. The association constant for formation of the hexamer from dimer and tetramer is lower by an order of magnitude than that for the association of two dimers. The association constant for the formation of the octamer from the hexamer is appreciably larger, and the value appears to reach a constant level for higher oligomers. These observations are explained in terms of conformational strain due to formation of cyclic structures, the distortion being greatest on passing from the tetramer to the hexamer. The association for a single-site interaction between the dimer and a univalent fragment has also been analyzed. The results show that the free energy generated by a single-point interaction is much greater than that obtained by averaging over all pairwise interactions within the oligomers, correcting for the effect of cratic entropy. The results are related to the association state of the spectrin prevailing in the cell. Phosphorylation at the physiological sites in the dimer does not appreciably change the thermodynamics of self-association, at least up to the hexamer.     

Structure of a loose dimer: an intermediate in nitric oxide synthase assembly

Cooperativity among ligand binding, subunit association, and protein folding has implications for enzyme regulation as well as protein aggregation events associated with disease. The binding of substrate l-arginine or cofactor tetrahydrobiopterin converts nitric oxide synthases (NOSs) from a "loose dimer", with an exposed active center and higher sensitivity to proteolysis, to a "tight dimer" competent for catalysis. The crystallographic structure of the Bacillus subtilis NOS loose dimer shows an altered association state with severely destabilized subdomains. Ligand binding or heme reduction converts loose dimers to tight dimers in solution and crystals. Mutations at key positions in the dimer interface that distinguish prokaryotic from eukaryotic NOSs affect the propensity to form loose dimers. The loose dimer structure indicates that non-native interactions can mediate subunit association in NOS.     

Thermal dissociation and unfolding of insulin

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

Identification of a ligand-binding region of the human insulin receptor encoded by the second exon of the gene

Structure-function studies of the insulin molecule indicate that an insulin B chain domain comprising residues 22-26 is involved both in binding to the insulin receptor (INSR) and in insulin dimer formation, suggesting that this domain might also interact with a structure resembling the insulin dimer interface in the INSR. Expression of a mutant INSR cDNA with a deletion of the region corresponding to exon 2 of the INSR gene produces a protein devoid of insulin-binding activity, although the mutant protein is processed appropriately to alpha- and beta-subunits, suggesting that the insulin-binding domain is encoded at least in part by exon 2. Within this region of the INSR molecule, the sequence 83-103 fulfills the structural criteria for a dimer interface. Studies of mutant INSRs with substitutions for phenylalanine 88 or 89 show that the presence of phenylalanine at position 89 is essential for full binding affinity.     

Gene 5 protein of bacteriophage fd: A dimer which interacts Co-operatively with DNA

Isolated gene 5 protein from bacteriophage fd-infected Escherichia coli has been shown by sedimentation equilibrium to exist primarily as a dimer under non-denaturing conditions. The dimer was stable under conditions of high ionic strength, extremes in pH, dilution to 0.075 mg/ml, and increased temperature. Gene 5 protein did not undergo the indefinite self-association observed with gene 32 protein.Three lines of evidence for co-operative binding of gene 5 protein to DNA were developed. First, the interaction between gene 5 protein and phage T4 DNA was examined using a nitrocellulose filter assay. Scatchard plots of the binding data indicated that the interaction was co-operative. Similar results were obtained with gene 32 protein. Second, the co-operative binding of both proteins to DNA was shown by the sensitivity of the protein-DNA interaction to increasing ionic strength at various ratios of protein to DNA. Finally, by using the cross-linking agent, dimethyl suberixmidate, oligomeric structures containing at least seven monomers were found when the DNA was less than saturated.The possibility that gene 5 protein dimers undergo indefinite self-association in the presence of oligonucleotides was examined by sedimentation equilibrium. With oligo[d(pT)₄], the protein dimer was complexed with this oligonucleotide but no self-association was observed. With oligo[d(pT)₈], gene 5 protein formed tetramers, but no significant indefinite association was noted. These results do not suggest a DNA-induced conformational change, which results in indefinite association. A model for the co-operative binding of gene 5 protein to DNA is presented.     

Munc 18-1 and granuphilin collaborate during insulin granule exocytosis

Munc 18-1 is a member of the Sec/Munc family of syntaxin-binding proteins known to bind to the plasma membrane Q-SNARE syntaxin1 and whose precise role in regulated exocytosis remains controversial. Here, we show that Munc 18-1 plays a positive role in regulated insulin secretion from pancreatic beta cells. Munc 18-1 depletion caused a loss in the secretory capacity of both transiently transfected INS 1E cells and a stable clone with tetracycline-regulated Munc 18-1 RNA interference. In addition, Munc 18-1-depleted cells exhibited defective docking of insulin granules to the plasma membrane and accumulated insulin in the trans Golgi network. Furthermore, glucose stimulation after Munc 18-1 depletion resulted in the rapid formation of autophagosomes. In contrast, overexpression of Munc 18-1 had no effect on insulin secretion. Although there was no detectable interaction between Munc 18-1 and Munc-18-interacting protein 1 or calcium/calmodulin-dependent serine protein kinase, Munc 18-1 associated with the granular protein granuphilin. This association was regulated by glucose and was required for the specific interaction of insulin granules with syntaxin1. We conclude that Munc 18-1 and granuphilin collaborate in the docking of insulin granules to the plasma membrane in an initial fusion-incompetent state, with Munc 18-1 subsequently playing a positive role in a later stage of insulin granule exocytosis.     

Deuterium NMR of 2HCO-Val1...gramicidin A and 2HCO-Val1-D-Leu2...gramicidin A in oriented DMPC bilayers.

Deuterium NMR is used to study the structure and dynamics of the formyl C-2H bond in selectively deuterated gramicidin molecules. Specifically, the functionally different analogues 2HCO-Val1...gramicidin A and 2HCO-Val1-D-Leu2...gramicidin A are studied by 2H NMR so that any conformational or dynamical differences between the two analogues can be correlated with their difference in lifetime. These analogues are first synthesized, purified, and characterized and then incorporated into oriented bilayers of dimyristoylphosphatidylcholine sandwiched between glass coverslips. Phosphorous NMR line shapes obtained from these samples are consistent with the presence of the bilayer phase and indicate that the disorder exhibited by the lipid matrix is approximately of the same type and degree for both analogues. Deuterium NMR line shapes obtained from these samples indicate that the motional axis of the formyl group of gramicidin is parallel to the coverslip normal, that the distribution of motional axis orientations has a width of 7-9 degrees, and that a similar, major conformational and dynamical state exists for the formyl C-2H bond of both analogues. In this state, if the only motion present is fast axial rotation, then the experimentally derived angle between the formyl C-2H bond and the motional axis is consistent with the presence of a right-handed, single-stranded, beta 6.3 helical dimer but is not consistent with the presence of a left-handed, single-stranded, beta 6.3 helical dimer. However, if fast axial rotation is not the only motion present, then the left-handed, single-stranded, beta 6.3 helical dimer cannot be absolutely excluded as a possibility. Also, a second, minor conformational and dynamical state appears to be present in the spectrum of 2HCO-Val1-D-Leu2...gramicidin A but is not observed in the spectrum of 2HCO-Val1...gramicidin A. This minor conformational and dynamical state may reflect the presence of monomers, while the major conformational and dynamical state may reflect the presence of dimers.     

The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation.

The allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T-state dimeric phosphorylase b and R-state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C-terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 A) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C-terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody-protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H-bonds per surface area (one H-bond per 130 A2). In addition, the surface area made inaccessible at the interface is relatively small (1,142 A2 per subunit on dimer to tetramer association compared with 2,217 A2 per subunit on monomer-to-dimer association).     

Suppression of insulin aggregation by heparin

The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH- and ionic strength ( I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH = pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I = 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.     

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 dynamics simulation of the C-terminal sterile alpha-motif domain of human p73alpha: evidence of a dynamical relationship between helices 3 and 5

We used molecular dynamics simulation to evaluate the association properties of C-terminal sterile alpha-motif (SAM) domain of human p73alpha. To test the dimerization propensity of this structure we carried out four simulations: EphB2 X-ray dimer, p73 modeled dimer, p73 NMR monomer, and p73 modeled monomer with an elongated helix 5. The results show a direct interaction between helix 5 and helix 3 since a conformational collapse of helix 3 is observed when dimer contact and/or an elongation of helix 5 is introduced by modeling in p73 SAM domain. On the basis of these results we suggest that the recognition properties of the SAM domains may be modulated by the conformational state of helix 5.     

Folding and oxidation of the antibody domain C(H)3

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge.