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.     

The Glu(B13) carboxylates of the insulin hexamer form a cage for Cd2+ and Ca2+ ions

Substitution of Cd2+ for Zn2+ yields a hexameric insulin species containing 3 mol of metal ion per hexamer. The Cd2+ binding loci consist of the two His(B10) sites and a new site involving the Glu(B13) residues located at the center of the hexamer [Sudmeier, J. L., Bell, S. J., Storm, M. C., & Dunn, M. F. (1981) Science (Washington, D.C.) 212, 560-562]. Substitution of Co2+ or Co3+ for Zn2+ gives hexamers containing 2 mol of metal per hexamer. Insulin solutions to which both Cd2+ and Co2+ have been added in a ratio of 6:2:1 [In]:[Co2+]:[Cd2+] followed by oxidation to the exchange-inert Co3+ state yield stable hybrid species containing both Co3+ and Cd2+ with a composition of (In)6(Co3+)2Cd2+. The kinetics of the reaction of 2,2',2"-terpyridine (terpy) with the exchange-labile (In)6(Cd2+)2 and (In)6(Co2+)2 derivatives are biphasic and involve the rapid formation of an intermediate with coordination of one terpy molecule to each protein-bound metal ion; then, in a rate-limiting step the terpy-coordinated metal ion dissociates from the protein, and a second molecule of terpy binds to the metal ion to form a bis complex. Reaction of the exchange-inert Co3+ ions of (In)6(Co3+)2 with terpy is a slow apparent first-order process (t1/2 = 13.1 h). In contrast to the kinetic behavior of (In)6(Co2+)2 and (In)6(Cd2+)2, the Cd2+ ions bound to the hybrid (In)6(Co3+)2Cd2+ react quite slowly with terpy (t1/2 = 1 h at pH 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Conformational analysis by nuclear magnetic resonance: insulin

High-resolution 270-MHz proton nuclear magnetic resonance (NMR) spectra of the native two-zinc insulin hexamer at pH 9 have been obtained, and assignments of key resonances have been made. Spectra of zinc-free insulin titrated with Zn2+ are unchanged after the addition of 1 equiv of zinc per insulin hexamer, indicating that the conformation of the hexamer is fixed at this point and that the second zinc ion does not significantly change the conformation. Titration of the two-zinc insulin hexamer with anions high on the Hofmeister series such as SCN- causes marked changes in the NMR spectra which are interpreted as the result of major conformational changes to a new hexameric form of insulin having a twofold axis perpendicular to the threefold axis. Analysis of difference spectra indicates that this new hexamer (which should be capable of binding six zinc ions) binds 2 equiv of SCN- at two sites which are assumed to be identical and independent (K1 = 10(3), K2 = 2.5 X 10(2) M-1).     

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.     

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.     

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.     

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.     

Structural consequences of the B5 histidine --> tyrosine mutation in human insulin characterized by X-ray crystallography and conformational analysis.

The addition of phenols to hexameric insulin solutions produces a particularly stable hexamer, resulting from a rearrangement in which residues B1-B8 change from an extended conformation (T-state) to form an alpha-helix (R-state). The R-state is, in part, stabilized by nonpolar interactions between the phenolic molecule and residue B5 His at the dimer-dimer interface. The B5 His --> Tyr mutant human insulin was constructed to see if the tyrosine side chain would mimic the effect of phenol binding in the hexamer and induce the R-state. In partial support of this hypothesis, the molecule crystallized as a half-helical hexamer (T(3)R(3)) in conditions that conventionally promote the fully nonhelical (T6) form. As expected, in the presence of phenol or resorcinol, the B5 Tyr hexamers adopt the fully helical (R6) conformation. Molecular modeling calculations were performed to investigate the conformational preference of the T-state B5 Tyr side chain in the T(3)R(3) form, this side chain being associated with structural perturbations of the A7-A10 loop in an adjacent hexamer. For an isolated dimer, several different orientations of the side chain were found, which were close in energy and readily interconvertible. In the crystal environment only one of these conformations remains low in energy; this conformation corresponds to that observed in the crystal structure. This suggests that packing constraints around residue B5 Tyr result in the observed structural rearrangements. Thus, rather than promoting the R-state in a manner analogous to phenol, the mutation appears to destabilize the T-state. These studies highlight the role of B5 His in determining hexamer conformation and in mediating crystal packing interactions, properties that are likely be important in vivo.     

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)     

Enhancing the T-->R transition of insulin by helix-promoting sequence modifications at the N-terminal B-chain

Structurally, the T-->R transition of insulin mainly consists of a rearrangement of the N-terminal B-chain (residues B1-B8) from extended to helical in one or both of the trimers of the hexamer. The dependence of the transition on the nature of the ligands inducing it, such as inorganic anions or phenolic compounds, as well as of the metal ions complexing the hexamer, has been the subject of extensive investigations. This study explores the effect of helix-enhancing modifications of the N-terminal B-chain sequence where the transition actually occurs, with special emphasis on N-capping. In total 15 different analogues were prepared by semisynthesis. 80% of the hexamers of the most successful analogues with zinc were found to adopt the T3R3 state in the absence of any transforming ligands, as compared to only 4% of wild-type insulin. Transformation with SCN- ions can exceed the T3R3 state where it stops in the case of wild-type insulin. Full transformation to the R6 state can be achieved by only one-tenth the phenol concentration required for wild-type insulin, i.e. almost at the stoichiometric ratio of 6 phenols per hexamer.     

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.     

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.     

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.     

Structural analysis of proinsulin hexamer assembly by hydroxyl radical footprinting and computational modeling

Mutations in the insulin gene can impair proinsulin folding and cause diabetes mellitus. Although crystal structures of insulin dimers and hexamers are well established, proinsulin is refractory to crystallization. Although an NMR structure of an engineered proinsulin monomer has been reported, structures of the wild-type monomer and hexamer remain undetermined. We have utilized hydroxyl radical footprinting and molecular modeling to characterize these structures. Differences between the footprints of insulin and proinsulin, defining a "shadow" of the connecting (C) domain, were employed to refine the model. Our results demonstrate that in its monomeric form, (i) proinsulin contains a native-like insulin moiety and (ii) the C-domain footprint resides within an adjoining segment (residues B23-B29) that is accessible to modification in insulin but not proinsulin. Corresponding oxidation rates were observed within core insulin moieties of insulin and proinsulin hexamers, suggesting that the proinsulin hexamer retains an A/B structure similar to that of insulin. Further similarities in rates of oxidation between the respective C-domains of proinsulin monomers and hexamers suggest that this loop in each case flexibly projects from an outer surface. Although dimerization or hexamer assembly would not be impaired, an ensemble of predicted C-domain positions would block hexamer-hexamer stacking as visualized in classical crystal lattices. We anticipate that protein footprinting in combination with modeling, as illustrated here, will enable comparative studies of diabetes-associated mutant proinsulins and their aberrant modes of aggregation.     

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.     

Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The solution structure of the B9(Asp) mutant of human insulin has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy. Thirty structures were calculated by distance geometry from 451 interproton distance restraints based on intra-residue, sequential and long-range nuclear Overhauser enhancement data, 17 restraints on phi torsional angles obtained from 3JH alpha HN coupling constants, and the restraints from 17 hydrogen bonds, and the three disulphide bridges. The distance geometry structures were optimized using restrained molecular dynamics (RMD) and energy minimization. The average root-mean-square deviation for the best 20 RMD refined structures is 2.26 A for the backbone and 3.14 A for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are better defined, with root-mean-square deviation values of 1.11 A for the backbone and 2.03 A for all atoms. The data analysis and the calculations show that B9(Asp) insulin, in water solution at the applied pH (1.8 to 1.9), is a well-defined dimer with no detectable difference between the two monomers. The association of the two monomers in the solution dimer is relatively loose as compared with the crystal dimer. The overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer is found to be preserved. The conformation-averaged NMR structures obtained for the monomer is close to the structure of molecule 1 in the hexamer of the 2Zn insulin crystal. However, minor, but significant deviations from this structure, as well as from the structure of monomeric insulin in solution, exist and are ascribed to the absence of the hexamer and crystal packing forces, and to the presence of monomer-monomer interactions, respectively. Thus, the monomer in the solution dimer shows a conformation similar to that of the crystal monomer in molecular regions close to the monomer-monomer interface, whereas it assumes a conformation similar to that of the solution structure of monomeric insulin in other regions, suggesting that B9(Asp) insulin adopts a monomer-like conformation when this is not inconsistent with the monomer-monomer arrangement in the dimer.     

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.     

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.