Spectroscopic signatures of the T to R conformational transition in the insulin hexamer

The cobalt(II)-substituted human insulin hexamer has been shown to undergo the phenol-induced T6 to R6 structural transition in solution. The accompanying octahedral to tetrahedral change in ligand field geometry of the cobalt ions results in dramatic changes in the visible region of the electronic spectrum and thus represents a useful spectroscopic method for studying the T to R transition. Changes in the Co2+ spectral envelope show that the aqua ligand associated with each tetrahedral Co2+ center can be replaced by SCN-, CN-, OCN-, N3-, Cl-, and NO2-. 19F NMR experiments show that the binding of m-trifluorocresol stabilizes the R6 state of zinc insulin. The chemical shift and line broadening of the CF3 singlet, which occur due to binding, provide a useful probe of the T6 to R6 transition. Due to the appearance of new resonances in the aromatic region, the 500 MHz 1H NMR spectrum of the phenol-induced R6 hexamer is readily distinguishable from that of the T6 form. 1H NMR studies show that phenol induces the T6 to R6 transition, both in the (GlnB13)6(Zn2+)2 hexamer and in the metal-free GlnB13 species; we conclude that metal binding is not a prerequisite for formation of the R state in this mutant.     

Structural transition in the metal-free hexamer of protein-engineered [B13 Gln]insulin

For hexamer formation of native insulin the repulsive potential of six B13 Glu carboxylate groups coming together in the centre is overcome by zinc binding to B10 His. Substitution of Gln for Glu in position B13 by site-directed mutagenesis, i.e. replacement of the repelling carboxylates by amide groups, which are offering H-bonding potential, enhances association and allows a metal-free hexamer to form. Merely upon addition of zinc ions this hexamer undergoes the T6----T3R3 respectively T6----R6 structural transition which in the native 2Zn insulin hexamer is inducible only by additives like inorganic anions or phenolic compounds. [B13 Gln]Insulin hexamers are transformed by phenolic compounds, but not by anions, even in the absence of any metal. The structural transformation of insulin can thus be brought about in two ways: By inorganic ions with the zinc ions as their points of attack, which preexist in the nontransformed hexamer, and by phenol, for which the binding sites close to the B5 histidines come into existence only with the transformation. Therefore transformed and non-transformed hexamers, i.e. molecules with helical and extended B chain N-terminus, must be related in a dynamic equilibrium. Phenol acts as a wedge jamming the structure in the transformed state and trapping the zinc ions. Combination of transformed 2Zn[B13 Gln]insulin and metal-free native insulin in the absence of additives results in a redistribution of the zinc ions in favour of native insulin which is an outcome of the dynamic equilibrium and also demonstrates an influence of B13 charge on metal binding affinity. Transformation of a single subunit in a hexamer would lead to bad contacts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer

The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-states which are further stabilized by binding of certain anions that do not stabilize the T6-state. It was shown that the thermostability of the resorcinol-stabilized R6-state was significantly higher than that of the T6-state. Further analysis showed that phenol- and m-cresol-stabilized R6-hexamer loses three ligands before reaching the unfolding temperature and hence unfolds from the T3R3-state. The relative affinity of the four tested anionic ligands was found, by DSC, to be thiocyanate > or = 4-hydroxy-3-nitrobenzoate > p-aminobenzoate > chloride. The results correlate with other methods and demonstrate that DSC provides a general and useful method of evaluation of both phenolic and anionic ligand binding to insulin without the use of probes or other alterations of the system of interest. However, it is a prerequisite that the binding is strong enough to saturate the binding sites at temperatures around the unfolding transition.     

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.     

The structure of a mutant insulin uncouples receptor binding from protein allostery. An electrostatic block to the TR transition

The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T(6), T(3)R(3)(f), and R(6). Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of His(B5) in wild-type structures: on the T(6) surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in His(B5) pK(a), in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of His(B5) (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of Arg(B5)-insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T(6) hexamers rather than R(6) as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3A, closely resembles the wild-type T(6) hexamer. Whereas Arg(B5) is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving His(B5) in wild-type T-states. The substantial receptor-binding activity of Arg(B5)-insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers.     

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.     

A solution equivalent of the 2Zn----4Zn transformation of insulin in the crystal

Circular dichroic spectroscopy clearly reveals a solvent-induced conformational change of insulin in the presence of zinc ions. The spectral change corresponds to an increase in helix content. The transition observed in solution is an equivalent of the 2Zn----4Zn insulin transformation in the crystal. This is inferred from a series of observations. (1) The spectral effects are compatible with the refolding of the B-chain N-terminus into a helix known from crystal studies. (2) The spectral effects are induced by the very same conditions which are known to induce the 2Zn----4Zn insulin transformation in the crystal (i.e. threshold concentrations of NaCl, KSCN, NaI, for example). (3) They fail to be induced by the same conditions that fail to induce the crystal transformation (e.g. Ni2+ instead of Zn2+). It is concluded that the potential to undergo the transition resides in the hexamer since neither insulin dimers nor monomeric des-pentapeptideB26-30-insulin respond detectably to high halide concentration. Secondly the ability of zinc ions to accommodate tetrahedral coordination allows the transition which is not permitted by other divalent metal ions. Thirdly the transition is independent of the off-axial tetrahedral zinc coordination sites since it occurs in [AlaB5]insulin which lacks the B5 histidine necessary for their formation. A symmetrically rearranged hexamer thus appears possible with two tetrahedrally coordinated zinc ions on the threefold axis; this is consistent with the observation that in native insulin two zinc ions per hexamer are sufficient to produce the full spectral effect. The amount of additional helix derived from the circular dichroic spectral change, however, cannot settle whether the transition comprises only three or all six of the subunits to yield a symmetrical hexamer. Finally the transformation in solution evidently still occurs in an intramolecularly A1-B29-cross-linked insulin in spite of the partially reduced flexibility.     

Metal induced structural changes observed in hexameric insulin

The metal ions in insulin hexamer play a crucial role in the T to R conformational transitions. We have determined the crystal structures of 2Mn2+, 1Rb1+ and 4Ni2+ human arg-insulin and compared them with the 2Zn2+ structure. The first two structures exist in the T3R3f state like the native 2Zn2+ arg-insulin, while the 4Ni2+ adopts a T6 conformation. The metal coordination is found to be tetrahedral in all the structures except that of nickel where a dual octahedral and tetrahedral coordination is found at one site. Rubidium occupies only one of the high affinity metal binding sites. The metal induced structural changes observed, have been explained.     

The T to R transition in the copper(II)-substituted insulin hexamer. Anion complexes of the R-state species exhibiting type 1 and type 2 spectral characteristics.

The R-state conformation of the Cu(II)-substituted insulin hexamer has been identified, and a number of its derivatives have been studied via 1H NMR, ESR, and UV-visible spectroscopy. This work establishes that the Cu(II)-substituted insulin hexamer undergoes an analogous T to R conformational transition in solution that has been identified previously for Zn(II)- and Co(II)-insulin hexamers [Roy, M., Brader, M.L., Lee, R. W.-K., Kaarsholm, N.C., Hansen, J., & Dunn, M.F. (1989) J. Biol. Chem. 264, 19081-19085]. The data indicate that each Cu(II) center of the R-state Cu(II)-insulin hexamer possesses a coordination site that is accessible to anions from solution. Both phenol and anionic ligands that coordinate to the Cu(II) ions are required to generate the necessary heterotropic interactions that stabilize the R-state structure. With phenylmethylthiolate (PMT), a Cu(II)-R6 adduct that displays the spectral features of blue (type 1) copper proteins is obtained. This complex is proposed to embody a pseudotetrahedral CuIIN3S(PMT) chromophore, in which N is HisB10 (imidazolyl). The remaining ligands examined gave rise to Cu(II)-R6 adducts that possessed the spectral characteristics of normal (type 2) Cu(II) proteins. Under reducing conditions, Cu(I)-T6 and Cu(I)-R6 hexamers have been identified.     

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].     

Cobalt probing of structural alternatives for insulin in solution

Inorganic anions and phenolic compounds make the subunits of insulin hexamers undergo the T----R transition whereby the extended N-terminal B chain becomes helical and the octahedral metal coordination tetrahedral. The role of the metal ions is permissive. With cresol the transition is also undergone by metal-free hexamers. For coordinative reasons only zinc insulin can be transformed by moderate concentrations of inorganic anions. At higher concentrations and particularly with cresol transformation is also possible if Zn2+ is replaced by other metal ions. Owing to its d--d transitions in the visible cobalt lends itself as a spectroscopic probe for studying the interdependence of transformation and coordination. The transformation-related change in coordination is reflected in both the isotropic absorption and the CD spectrum. Cresol achieves T6----R6 transformation whereas that induced by SCN- ions is T6----T'3R3 with only the axial metal-binding site being realized in the R3 trimer. The spectral effects of the transformation of the two trimers are not additive; an extra contribution seems to be indicative of trimer/trimer interaction. Oxidation of 2 Co2+ insulin to a certain extent affects the structure of insulin; a characteristic positive band appears at 251 nm. Because of its extremely stable and exclusively octahedral complexes the Co3+ ion most strongly withstands transformation. The oxidation of tetrahedrally liganded Co2+ ions in R3 trimers proceeds with reduced velocity. Independent transformation of the Zn2+ trimers is possible in Zn2+/Co3+ metal hybrids of insulin.     

Structural signatures of the complex formed between 3-nitro-4-hydroxybenzoate and the Zn(II)-substituted R(6) insulin hexamer

3-Nitro-4-hydroxybenzoate (3N4H) is a probe of the structure and dynamics of the metal-centered His B10 assembly sites of the insulin hexamer. Each His B10 site consists of a approximately 12 A-long cavity situated on the threefold symmetry axis. These sites play an important role in the storage and release of insulin in vivo. The allosteric behavior of the insulin hexamer is modulated by ligand binding to the His B10 zinc sites and to the phenolic pockets. Binding to these sites drives transitions among three allosteric states, designated T(6), T(3)R(3), and R(6). Although a wide variety of mono anions bind to the His B10 zinc sites of R(3), X-ray structures of ligands complexed to this site exist only for H(2)O, Cl(-), and SCN(-). This work combines one- and two-dimensional (1)H NMR and UV-Vis absorbance studies of the structure and dynamics of the 3N4H complex, which establish the following: (1). relative to the NMR time scale, 3N4H exchange between free and bound states is slow, while flipping among three equivalent orientations about the site threefold axis is fast; (2). binding of 3N4H perturbs resonances within the His B10 zinc site and generates NOEs between ligand resonances and the insulin C-alpha and side chain resonances of ValB2, AsnB3, LeuB6, and CysB7; and (3).3N4H exchange for other ligands is limited by a protein conformational transition. These results are consistent with coordination of the 3N4H carboxylate to the His B10 zinc ion and van der Waals interactions with Val B2, Asn B3, Leu B6, and Cys A7.     

Kinetic measurements of T----R structural transitions in insulin.

The T6----T3R3 and T3R3----R6-structural transitions of cobalt insulin hexamers as induced by SCN ions or m-cresol were studied in stopped-flow experiments using the absorption in the visible for monitoring their time course. The T6----T3R3 transition induced by either SCN or limited concentrations of m-cresol is mono-exponential with a rate constant of 0.1 s-1 and 0.4 s-1, respectively. A mono-exponential time course is also encountered for the m-cresol-induced T3R3----R6 transition when starting from the T3R3 state preestablished by either SCN or m-cresol. The corresponding rate constants are 1.3 s-1 and 0.49 s-1, respectively. If m-cresol is used beyond the concentration range where transformation is limited to one trimer, two exponentials are required for fitting the time course. The second exponential corresponds to the T3R3----R6 step with a concentration-independent rate constant of 0.4 s-1. The rate constant for the faster T6----T3R3 transition, however, increases with increasing excess of m-cresol.     

胰岛素六聚体在水溶液中的构象柔性研究

用分子动力学（MD)模拟方法设计了两个模拟时间为600ps的对比计算机模拟实验,研究了R6态的胰岛素六聚体在水溶液中的构象柔性。通过对MD模拟所得到的轨迹的分析发现,包含锌离子和苯酚的胰岛素六聚体体系的构象柔性弱于不含锌离子和苯酚的胰岛素六聚体体系,对于不包含锌离子和苯酚的体系,胰岛素六聚体的构象柔性表现得较为突出,特别是在实验研究认为与胰岛素和受体结合位点有关的每个单体的B链羧端的β折叠部分,发生了快速而显著的构象变化,表现出了很大的构象柔性。这些模拟结果与实验观测结果相吻合。     

Structural effects of protein lipidation as revealed by LysB29-myristoyl, des(B30) insulin

Intracellular proteins are frequently modified by covalent addition of lipid moieties such as myristate. Although a functional role of protein lipidation is implicated in diverse biological processes, only a few examples exist where the structural basis for the phenomena is known. We employ the insulin molecule as a model to evaluate the detailed structural effects induced by myristoylation. Several lines of investigation are used to characterize the solution properties of Lys(B29)(N(epsilon)-myristoyl) des(B30) insulin. The structure of the polypeptide chains remains essentially unchanged by the modification. However, the flexible positions taken up by the hydrocarbon chain selectively modify key structural properties. In the insulin monomer, the myristoyl moiety binds in the dimer interface and modulates protein-protein recognition events involved in insulin dimer formation and receptor binding. Myristoylation also contributes stability expressed as an 30% increase in the free energy of unfolding of the protein. Addition of two Zn(2+)/hexamer and phenol results in the displacement of the myristoyl moiety from the dimer interface and formation of stable R(6) hexamers similar to those formed by human insulin. However, in its new position on the surface of the hexamer, the fatty acid chain affects the equilibria of the phenol-induced interconversions between the T(6), T(3)R(3), and R(6) allosteric states of the insulin hexamer. We conclude that insulin is an attractive model system for analyzing the diverse structural effects induced by lipidation of a compact globular protein.     

Role of metal ions in the T- to R-allosteric transition in the insulin hexamer.

The role of metal ions in the T- to R-allosteric transition is ascertained from the investigation of the T- to R-allosteric transition of transition metal ions substituted-insulin hexamers, as well as from the kinetics of their dissociation. These studies establish that ligand field stabilization energy (LFSE), coordination geometry preference, and the Lewis acidity of the metal ion in the zinc sites modulate the T- to R-state transition. (1)H NMR, (113)Cd NMR, and UV-vis measurements demonstrate that, under suitable conditions, Fe2+/3+, Ni2+, and Cd2+ bind insulin to form stable hexamers, which are allosteric species. (1)H NMR R-state signatures are elicited by addition of phenol alone in the case of Ni(II)- and Cd(II)-substituted insulin hexamers. The Fe(II)-substituted insulin hexamer is converted to the ferric analogue upon addition of phenol. For the Fe(III)-substituted insulin hexamer, appearance of (1)H NMR R-state signatures requires, additionally to phenol, ligands containing a nitrogen that can donate a lone pair of electrons. This is consistent with stabilization of the R-state by heterotropic interactions between the phenol-binding pocket and ligand binding to Fe(III) in the zinc site. UV-vis measurements indicate that the (1)H NMR detected changes in the conformation of the Fe(III)-insulin hexamer are accompanied by a change in the electronic structure of the iron site. Kinetic measurements of the dissociation of the hexamers provide evidence for the modulation of the stability of the hexamer by ligand field stabilization effects. These kinetic studies also demonstrate that the T- to R-state transition in the insulin hexamer is governed by coordination geometry preference of the metal ion in the zinc site and the compatibility between Lewis acidity of the metal ion in the zinc site and the Lewis basicity of the exogenous ligands. Evidence for the alteration of the calcium site has been obtained from (113)Cd NMR measurements. This finding adds to the number of known conformational changes that occur during the T- to R-transition and is an important consideration in the formulation of allosteric mechanisms of the insulin hexamer.     

Toward the insulin-IGF-I intermediate structures: functional and structural properties of the [TyrB25NMePheB26] insulin mutant

The origins of differentiation of insulin from insulin-like growth factor I (IGF-I) are still unknown. To address the problem of a structural and biological switch from the mostly metabolic hormonal activity of insulin to the predominant growth factor activities of IGF-I, an insulin analogue with IGF-I-like structural features has been synthesized. Insulin residues Phe(B25) and Tyr(B26) have been swapped with the IGF-I-like Tyr(24) and Phe(25) sequence with a simultaneous methylation of the peptide nitrogen of residue Phe(B26). These modifications were expected to introduce a substantial kink in the main chain, as observed at residue Phe(25) in the IGF-I crystal structure. These alterations should provide insight into the structural origins of insulin-IGF-I structural and functional divergence. The [Tyr(B25)NMePhe(B26)] mutant has been characterized, and its crystal structure has been determined. Surprisingly, all of these changes are well accommodated within an insulin R6 hexamer. Only one molecule of each dimer in the hexamer responds to the structural alterations, the other remaining very similar to wild-type insulin. All alterations, modest in their scale, cumulate in the C-terminal part of the B-chain (residues B23-B30), which moves toward the core of the insulin molecule and is associated with a significant shift of the A1 helix toward the C-terminus of the B-chain. These changes do not produce the expected bend of the main chain, but the fold of the mutant does reflect some structural characteristics of IGF-1, and in addition establishes the CO(A19)-NH(B25) hydrogen bond, which is normally characteristic of T-state insulin.     

Insulin hexamers: new conformations and applications

Recent studies on the structural and chemical properties of insulin have shown that the insulin hexamer is an allosteric protein capable of adopting three distinct conformations, designated T6, T3R3 and R6. Although the physiological consequences of this allostery are not established, new applications for the insulin hexamer as a model system for the study of allostery and for the study of zinc enzymes and copper proteins are emerging.     

The R-state proinsulin and insulin hexamers mimic the carbonic anhydrase active site

The cobalt(II)-substituted proinsulin and insulin hexamers have been studied in solution via electronic absorption spectroscopy. Hexameric proinsulin is shown to undergo the phenol-induced T6 to R6 conformational transition in a manner analogous to that previously established for insulin. In the absence of coordinating anions, the coordination spheres of the Co(II) ions in the proinsulin and insulin R6 hexamers comprise identical pseudotetrahedral arrangements of 3 histidine residues and 1 hydroxide ion. At alkaline pH, the visible absorption spectrum of the phenol-induced R6 Co(II) center is strikingly similar to the distinctive spectrum of the alkaline form of Co(II)-carbonic anhydrase. Exogenous ligands may coordinate to the Co(II) ions of the R6 proinsulin and insulin hexamers via replacement of the hydroxide ion, forming pseudotetrahedral adducts possessing characteristic spectra. The binding affinity of such ligands is shown to be strongly pH-dependent. The data presented establish that, although the Co(II)-substituted proinsulin and insulin R6 hexamers lack enzyme-like activity, these species duplicate spectrochemical characteristics of the Co(II)-carbonic anhydrase active site that are believed to be important signatures of carbonic anhydrase catalytic function.