X-ray structure of an unusual Ca2+ site and the roles of Zn2+ and Ca2+ in the assembly, stability, and storage of the insulin hexamer.

Metal ion binding to the insulin hexamer has been investigated by crystallographic analysis. Cadmium, lead, and metal-free hexamers have been refined to R values of 0.181, 0.172, and 0.172, against data of 1.9-, 2.5-, and 2.5-A resolution, respectively. These structures have been compared with each other and with the isomorphous two-zinc insulin. The structure of the metal-free hexamer shows that the His(B10) imidazole rings are arranged in a preformed site that binds a water molecule and is poised for Zn2+ coordination. The structure of the cadmium derivative shows that the binding of Cd2+ at the center of the hexamer is unusual. There are three symmetry-related sites located within 2.7 A of each other, and this position is evidently one-third occupied. It is also shown that the coordinating B13 glutamate side chains of this derivative have two partially occupied conformations. One of these conformations is two-thirds occupied and is very similar to that seen in two-zinc insulin. The other, one-third-occupied conformation, is seen to coordinate the one-third-occupied metal ion. The binding of Ca2+ to insulin is assumed to be essentially identical with that of Cd2+. Thus, we conclude that the Ca2+ binding site in the insulin hexamer is unlike that of any other known calcium binding protein. The crystal structures reported herein explain how binding of metal ions stabilizes the insulin hexamer. The role of metal ions in hexamer assembly and dissociation is discussed.     

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.     

Comparison of solution structural flexibility and zinc binding domains for insulin, proinsulin, and miniproinsulin

The chromophoric divalent metal ion chelators 4-(2-pyridylazo)resorcinol (PAR) and 2,2',2"-terpyridine (terpy) are used as kinetic and spectroscopic probes to investigate in solution the SCN- -induced conformational transformations of the insulin, proinsulin, and miniproinsulin hexamers (miniproinsulin is a proinsulin analogue wherein the C-chain is replaced by a dipeptide cross-link between Gly-A1 and Ala-B30). Herein we designate the 2Zn and 4Zn crystal forms of the hexamer as the T6 and T3R3 conformations, respectively. For all three proteins, addition of SCN- reduces the rate of sequestering and removal of zinc ion by chelator. The effect of SCN- on the rate of this process saturates at the same concentration (30 mM) known to induce the T6 to T3R3 transformation in the insulin crystal. Under both T6 and T3R3 conditions, the critical stoichiometry for high-affinity interaction between Zn2+ and each of the three proteins is shown to be 2 mol of Zn2+/mol of protein hexamer. Consequently, we confirm the finding that off-axial coordination of Zn2+ via His-B10 and His-B5 residues is of minor importance for the SCN- -induced conformation change in solution [Renscheidt, H., Strassburger, W., Glatter, U., Wollmer, A., Dodson, G. G., & Mercola, D. A. (1984) Eur. J. Biochem. 142, 7-14]. Under T6 conditions, the kinetics of the reactions between insulin, proinsulin, and miniproinsulin and a variable excess of terpy are similar and biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)     

500-MHz1H NMR studies of insulin: Complete assignment of histidine resonances

The two histidines of the insulin monomer play a vital role in the organization of insulin into insulin hexamers. The B10 histidines bind to zinc to form two-zinc insulin hexamer, and both the B5 and B10 histidines are implicated in the formation of four-zinc insulin hexamer. These two histidines are both accessible to solvent in the dimeric form of insulin, the predominant species present at pH 2–3. In the present work we report the first 500-MHz¹H NMR studies of insulin. At this frequency all four proton resonances from the two histidines of each equivalent monomer are resolved. The resonances are assigned to the C(2)- and C(4)-imidazole protons of B5 His and B10 His employing Carr-Purcell pulse sequences to detect singlets and to observe approximateT ₂ relaxation times. Zinc-free bovine insulin at pH 2.9 was examined at temperatures up to 60°C in acetate buffer and in urea of varying concentrations. The environments of B5 His in molecule I and molecule II of the dimer must be the same, with the same being true for B10 His, since a total of only four sharp resonances are seen. Our assignments for the two C(2) protons are consistent with those determined from recent studies of human (B5 Ala) insulin.     

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.     

Effects of calcium ion on ternary complexes formed between 4-(2-pyridylazo)resorcinol and the two-zinc insulin hexamer

As a means for probing the microenvironment of zinc in the insulin hexamer and to investigate the effects of calcium ion on the assembly and the structure of the two-zinc insulin hexamer, the thermodynamics and kinetics of the reaction between the chromophoric divalent metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) and zinc-insulin have been investigated over a wide range of conditions. For [PAR]0 much greater than [Zn2+]0 and [Zn2+]/[In] less than or equal to 0.33, the reaction leads to the sequestering and ultimate removal of all of the insulin-bound Zn2+; for [Zn2+]0 much greater than [PAR]0, two stable ternary complexes are formed where Zn2+ has ligands derived from PAR as well as from hexameric insulin. For [Zn2+]/[In] ratios below 0.33, the equilibrium distribution between the two ternary complexes is dependent on the [Zn2+]/[In] ratio. One of the complexes is assigned to the monoanion of PAR coordinated to Zn2+ that resides in a His-B10 site. The other complex is proposed to involve the coordination of (PAR)Zn to the site formed by the alpha-NH2 group of Phe-B1 and the gamma-carboxylate ion of Glu-A17 across the dimer-dimer interface on the surface of the hexamer. With either PAR or zinc-insulin in large excess, the kinetics of the PAR optical density changes are remarkably similar and biphasic. The faster step is first order in PAR and first order in insulin-bound Zn2+ (k congruent to 3 X 10(3) M-1 s-1) and involves the formation of an intermediate in which PAR is coordinated to insulin-bound zinc at the His-B10 site.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.     

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.     

Characterization of the R-state insulin hexamer and its derivatives. The hexamer is stabilized by heterotropic ligand binding interactions

1H NMR and UV-visible electronic absorption studies have been performed to investigate the effects of anions and cyclic organic molecules on the interconversion of the T- and R-conformational states (Kaarsholm et al., 1989) of hexameric M (II)-substituted insulin in solution (M = Zn or Co.). Two ligand binding processes that stabilize the R-state conformation of the M(II)-substituted insulin hexamer [M(II)-R6] have been distinguished: (i) The binding of neutral organic molecules to the six, crystallographically identified, protein pockets in the Zn(II)-R6 insulin hexamer (Derewenda et al. 1989) generate homotropic site-site interactions that stabilize the R-state. Cyclohexanol, phenol, 4-nitrophenol, and 4-hydroxymethylbenzoate are shown to bind at these sites. (ii) The coordination of singly charged anions that are able to gain access to the two HisB10 coordinated metal ions of the M(II)-R6 hexamer stabilizes the R-state. Adducts of the M(II)-R6 hexamer are formed, thereby, in which the solvent-accessible fourth coordination position of the M(II) ion is replaced by a competing anion. Binding to these two classes of sites introduces strong heterotropic interactions that stabilize the R-state. UV-visible spectral data and apparent affinity constants for the adducts formed by the Co(II)-R6 hexamer with a wide range of anionic ligands are presented. The Co(II)-R6 adducts have a strong preference for the formation of pseudotetrahedral Co(II) centers. The HCO3- and pyridine-2-thiolate ions form Co(II)-R6 adducts that are proposed to possess pentacoordinate Co(II) geometries. The relevance of the Co(II)-R6 complexes to carbonic anhydrase catalysis and zinc enzyme model systems is discussed.     

1H n.m.r. studies of insulin. Reversible transformation of 2-zinc to 4-zinc insulin hexamer

1H n.m.r. studies at 270 MHz were made of the transformation of 2 Zn insulin hexamer to 4 Zn hexamer produced by the addition of anions (thiocyanate ion). Four separate H2 histidine resonances were observed for the B5 and B10 histidines in 2 Zn hexamer at pH 7 and 9 and four separate resonances also occurred in the 4 Zn hexamer. The observation of these resonances and others from phenylalanine, tyrosine and leucine residues showed that the 2 Zn to 4 Zn transformation probably occurred in solution in a similar manner to that observed in the crystal. Furthermore as occurred in the crystal, it was found that in solution the transformation was reversible (on removal of thiocyanate) and that 2 Cd insulin was unable to undergo the transformation. Des-Phe-Bl-insulin did not undergo the transformation. Addition of SCN- to Zn-free insulin (mainly dimer) produced only a small transformation, consistent with the idea that Zn2+ promotes formation of hexamer from dimer but probably does not otherwise affect the transformation.     

Role of B13 Glu in insulin assembly. The hexamer structure of recombinant mutant (B13 Glu-->Gln) insulin.

The assembly of the insulin hexamer brings the six B13 glutamate side-chains at the centre into close proximity. Their mutual repulsion is unfavourable and zinc co-ordination to B10 histidine is necessary to stabilize the well known zinc-containing hexamers. Since B13 is always a carboxylic acid in all known sequences of hexamer forming insulins, it is likely to be important in the hormone's biology. The mutation of B13 Glu-->Gln leads to a stable zinc-free hexamer with somewhat reduced potency. The structures of the zinc-free B13 Gln hexamer and the 2Zn B13 insulin hexamer have been determined by X-ray analysis and refined with 2.5 A and 2.0 A diffraction data, respectively. Comparisons show that in 2Zn B13 Gln insulin, the hexamer structure (T6) is very like that of the native hormone. On the other hand, the zinc-free hexamer assumes a quaternary structure (T3/R3) seen in the native 4Zn insulin hexamer, and normally associated only with high chloride ion concentrations in the medium. The crystal structures show the B13 Gln side-chains only contact water in contrast to the B13 glutamate in 2Zn insulin. The solvation of the B13 Gln may be associated with this residue favouring helix at B1 to B8. The low potency of the B13 Gln insulin also suggests the residue influences the hormone's conformation.     

Characterization of the internal calcium(II) binding sites in dissolved insulin hexamer using europium(III) fluorescence

The fluorescence of Eu(III) is used to study the nature of the Ca(II) binding sites in the central cavity of the two-zinc(II) insulin hexamer. The dependence of the Eu(III) fluorescence lifetime upon Eu(III) stoichiometry indicates that there are three identical Eu(III) binding sites present in the two-zinc(II) insulin hexamer in solution. Addition of excess Ca(II) causes a decrease in the Eu(III) fluorescence intensity, confirming that Ca(II) competes for the observed Eu(III) sites. The solvent dependence of the Eu(III) fluorescence lifetime (H2O vs. D2O) indicates that four OH groups are coordinated to each Eu(III) in the hexamer. Substitution of Co(II) for Zn(II) causes a decrease in the Eu(III) fluorescence lifetime. Calculations based on F?rster energy-transfer theory predict that the Co(II) [or Zn(II) in vivo] and Eu(III) [or Ca(II) in vivo] binding sites are separated by 9.6 +/- 0.5 A. Variation of the metal stoichiometries indicates that all three Eu(III) [or Ca(II) in vivo] sites are equidistant from the Zn(II) sites. We conclude that these sites are identical with the three central Zn(II) sites present in insulin hexamer crystals soaked in excess Zn(II) [Emdin, S. O., Dodson, G., Cutfield, J. M., & Cutfield, S. M. (1980) Diabetologia 19, 174-182] and suggest that these central sites are occupied by Ca(II) in vivo.     

Crystal structure of allo-Ile(A2)-insulin, an inactive chiral analogue: implications for the mechanism of receptor binding

The crystal structure of an inactive chiral analogue of insulin containing nonstandard substitution allo-Ile(A2) is described at 2.0 A resolution. In native insulin, the invariant Ile(A2) side chain anchors the N-terminal alpha-helix of the A-chain to the hydrophobic core. The structure of the variant protein was determined by molecular replacement as a T(3)R(3) zinc hexamer. Whereas respective T- and R-state main-chain structures are similar to those of native insulin (main-chain root-mean-square deviations (RMSD) of 0.45 and 0.54 A, respectively), differences in core packing are observed near the variant side chain. The R-state core resembles that of the native R-state with a local inversion of A2 orientation (core side chain RMSD 0.75 A excluding A2); in the T-state, allo-Ile(A2) exhibits an altered conformation in association with the reorganization of the surrounding side chains (RMSD 0.98 A). Surprisingly, the core of the R-state is similar to that observed in solution nuclear magnetic resonance (NMR) studies of an engineered T-like monomer containing the same chiral substitution (allo-Ile(A2)-DKP-insulin; Xu, B., Hua, Q. X., Nakagawa, S. H., Jia, W., Chu, Y. C., Katsoyannis, P. G., and Weiss, M. A. (2002) J. Mol. Biol. 316, 435-441). Simulation of NOESY spectra based on crystallographic protomers enables the analysis of similarities and differences in solution. The different responses of the T- and R-state cores to chiral perturbation illustrates both their intrinsic plasticity and constraints imposed by hexamer assembly. Although variant T- and R-protomers retain nativelike protein surfaces, the receptor-binding activity of allo-Ile(A2)-insulin is low (2% relative to native insulin). This seeming paradox suggests that insulin undergoes a change in conformation to expose Ile(A2) at the hormone-receptor interface.     

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.     

Anion binding to neutral and positively charged lipid membranes

Aqueous anion binding to bilayer membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated by using deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Only those anions that exhibit chaotropic properties showed significant binding to POPC membranes. A detailed investigation of thiocyanate binding to neutral POPC and to positively charged mixed POPC/dihexadecyldimethylammonium bromide (DHDMAB) (8:2 mol/mol) membranes revealed changes in the 2H NMR quadrupole splittings from POPC specifically deuteriated at either the alpha-segment or the beta-segment of the choline head group which were consistent with a progressive accumulation of excess negative charge at the membrane surface with increasing SCN- concentration. Both the 2H and 31P NMR spectra indicated the presence of fluid lipids in a bilayer configuration up to at least 1.0 M NaSCN with no indication of any phase separation of lipid domains. Calibration of the relationship between the change in the 2H NMR quadrupole splitting and the amount of SCN- binding provided thiocyanate binding isotherms. At a given SCN- concentration the positively charged membranes bound levels of SCN- 3 times that of the neutral membranes. The binding isotherms were analyzed by considering both the electrostatic and the chemical equilibrium contributions to SCN- binding. Electrostatic considerations were accounted for by using the Gouy-Chapman theory. For 100% POPC membranes as well as for mixed POPC/DHDMAB (8:2 mol/mol) membranes the thiocyanate binding up to concentrations of 100 mM was characterized by a partition equilibrium with an association constant of K approximately 1.4 +/- 0.3 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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].     

Differences in the nature of the interaction of insulin and proinsulin with zinc

1. The reversible interaction of zinc with pig insulin and proinsulin has been studied at pH7 by equilibrium dialysis (ultrafiltration) and by sedimentation equilibrium and velocity measurements in the ultracentrifuge. Binding values calculated from equilibria, where the ratio of free to bound zinc was varied in the range 0.01:1-10:1, indicated that proinsulin and insulin each contained two main orders of zinc binding with very different affinities for the metal. 2. In equilibria containing low concentrations of free zinc (free: bound ratios of 0.01-0.1:1) both insulin and proinsulin aggregated to form soluble hexamers containing firmly bound zinc (up to 0.284g-atom/monomer) with an apparent intrinsic association constant of 1.9x10(6)m(-1). 3. Higher concentrations of zinc (free: bound ratios of 0.1-10.0:1) resulted in a progressive difference in the zinc binding, aggregation and solubility properties of the metal complexes of insulin and proinsulin. At the highest concentration of free zinc, proinsulin bound a total of more than 5.0g-atom/monomer and aggregated to form a mixture of soluble polymers (mainly 5.1S). In contrast, insulin bound a total of only 1.0g-atom/monomer and was almost completely precipitated from solution. 4. These results would indicate that the presence of the peptide segment connecting the insulin moiety in proinsulin does not prevent the firm binding of zinc to the insulin moiety and the formation of hexamers of zinc-proinsulin. At the same time although the connecting peptide contains additional sites of lower affinity for zinc, which should facilitate inter- and intra-molecular cross-linking, the general conformation of the zinc-proinsulin hexamer must preclude the formation of very large and close-packed aggregates that are insoluble in solutions at equilibrium.     

An electron paramagnetic resonance study of native and modified freeze-dried cupric insulin hexamer.

Cupric insulin was modified by the addition of cross-linking disulphide bridges between hexamers. The electron paramagnetic resonance (EPR) spectrum of this freeze-dried material was compared with that of freeze-dried unmodified cupric insulin containing various amounts of copper and added water. The modified insulin was found to have cupric ion sites magnetically very similar to that of native insulin containing two cupric ions per hexamer. Native hexamer produced in the presence of 2 Cu(II) ions per hexamer gave, after freeze-drying, an EPR spectrum with A_∥^Cu=16.5 mT, g_∥=2.285 and g_⊥=2.059 (site 1). The use of 4 or 6 Cu(II) ions per hexamer resulted in spectra with two components-a major component with the same A_∥^Cu and g_∥ values as the sample containing 2 Cu(II) ions (site 1) and an additional minor component (site 2). These sites have been identified with the analogous zinc binding site within the hexamer formed by three B-10 histidine residues (site 1) [1, 2] and the site formed by the B-1 α-amino and A-17 glutamyl-γ-barboxylic acid functions where excess zinc is bound (site 2) [3, 4]. The addition of water to native hexamer containing 2, 4, or 6 Cu(II) ions resulted in the appearance of three distinct EPR absorptions, one of which had the same parameters as the freeze-dried native insulin containing 2 Cu(II) ions per hexamer (site 1). Two further sites appeared (3 and 4) with the following parameters: A_∥^Cu=15.0 mT, g_∥=2.353, and g_⊥=2.07; A_∥^Cu=16.5 mT, g_∥=2.315, and g_⊥=2.07, respectively.     

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.     

Metal induced conformational changes in human insulin: crystal structures of Sr2+, Ni2+ and Cu2+ complexes of human insulin

Crystal structures of Sr(2+), Ni(2+) and Cu(2+) of human insulin complexes have been determined. The structures of Sr(2+) and Ni(2+) complexes are similar to Zn(2+) insulin and are in T6 conformation. (All the six monomers in the insulin hexamer are in Tensed conformation (T), which means the first eight residues of B-chain are in an extended conformation). Cu(2+) complex, though it assumes T6 conformation, has more structural differences due to lowering of crystal symmetry and space group shift from H3 (Hexagonal crystal system) to P3 (Trigonal crystal system) and a doubling of the c axis. 2Ni(2+) human insulin when compared to 4Ni(2+) Arg insulin suggests that terminal modifications may be responsible for additional metal binding. All the three metals have been shown to have a role in diabetes and hence may be therapeutically useful.