Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis

The presence of the copper ion at the active site of human wild type copper-zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyze the disproportionation of superoxide into dioxygen and hydrogen peroxide. Wild type CuZnSOD and several of the mutants associated with familial amyotrophic lateral sclerosis (FALS) (Ala(4) --> Val, Gly(93) --> Ala, and Leu(38) --> Val) were expressed in Saccharomyces cerevisiae. Purified metal-free (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification studies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis. From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in their proper locations in vitro. Similar studies of the wild type and FALS mutant CuZnSOD holoenzymes in the "as isolated" metallation state showed abnormally low copper-to-zinc ratios, although all of the copper acquired was located at the native copper binding sites. Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS). The loss of metal ion binding specificity of FALS mutant CuZnSODs in vitro may be related to their role in ALS.     

Crystallographic structures of bovine copper-zinc superoxide dismutase reveal asymmetry in two subunits: functionally important three and five coordinate copper sites captured in the same crystal

A key feature of the generally accepted catalytic mechanism of CuZn superoxide dismutase (CuZnSOD) is the breakage of the imidazolate bridge between copper and zinc and the loss of a coordinated water molecule from copper on reduction from Cu(II) to Cu(I). Crystal structures exist for the enzyme from a number of sources in the oxidised, five coordinate copper form. For the reduced form two structures from different sources have been determined only recently but provide contradictory results. We present crystal structures of bovine CuZnSOD (BSOD) in two different space groups. The structure of the P212121 form (pBSOD), at 1.65 A resolution clearly shows one subunit with Cu in the five coordinate, oxidised form, and the other with Cu in the three coordinate form expected for the reduced state. This mixed state of pBSOD is confirmed by XANES data of these crystals. The pBSOD structure has thus captured each subunit in one of the two oxidation state conformations and thus provides direct crystallographic evidence for the superoxide dismutase mechanism involving the breakage of the imidazole bridge between Cu and Zn. A shift in the position of copper in subunit A poises the catalytic centre to undergo the first stage of catalysis via dissociation of Cu from His61 with a concomittant movement of the coordinated water molecule towards His61, which rotates by approximately 20 degrees, enabling it to form a hydrogen bond to the water molecule. The Cu-Zn separation in the reduced site is increased by approximately 0.5 A. In contrast the 2.3 A resolution structure in space group C2221 (cBSOD) shows both of the Cu atoms to be in the five coordinate, oxidised form but in this space group the whole of subunit A is significantly more disordered than subunit B. An examination of published structures of "oxidised" SODs, shows a trend towards longer Cu-Zn and Cu-His61 separations in subunit A, which together with the structures reported here indicate a potential functional asymmetry between the subunits of CuZnSODs. We also suggest that the increased separation between Cu and Zn is a precursor to breakage of His61.     

The solution structure of reduced dimeric copper zinc superoxide dismutase. The structural effects of dimerization.

The solution structure of homodimeric Cu2Zn2 superoxide dismutase (SOD) of 306 aminoacids was determined on a 13C, 15N and 70% 2H labeled sample. Two-thousand eight-hundred and five meaningful NOEs were used, of which 96 intersubunit, and 115 dihedral angles provided a family of 30 conformers with an rmsd from the average of 0.78 +/- 0.11 and 1.15 +/- 0.09 A for the backbone and heavy atoms, respectively. When the rmsd is calculated for each subunit, the values drop to 0.65 +/- 0.09 and 1.08 +/- 0.11 A for the backbone and heavy atoms, respectively. The two subunits are identical on the NMR time scale, at variance with the X-ray structures that show structural differences between the two subunits as well as between different molecules in the unit cell. The elements of secondary structure, i.e. eight beta sheets, are the same as in the X-ray structures and are well defined. The odd loops (I, III and V) are well resolved as well as loop II located at the subunit interface. On the contrary, loops IV and VI show some disorder. The residues of the active cavity are well defined whereas within the various subunits of the X-ray structure some are disordered or display different orientation in different X-ray structure determinations. The copper(I) ion and its ligands are well defined. This structure thus represents a well defined model in solution relevant for structure-function analysis of the protein. The comparison between the solution structure of monomeric mutants and the present structure shows that the subunit-subunit interactions increase the order in loop II. This has the consequences of inducing the structural and dynamic properties that are optimal for the enzymatic function of the wild-type enzyme. The regions 37-43 and 89-95, constituting loops III and V and the initial part of the beta barrel and showing several mutations in familial amyotrophis lateral sclerosis (FALS)-related proteins have a quite extensive network of H-bonds that may account for their low mobility. Finally, the conformation of the key Arg143 residue is compared to that in the other dimeric and monomeric structures as well as in the recently reported structure of the CCS-superoxide dismutase (SOD) complex.     

The perplexing role of copper-zinc superoxide dismutase in amyotrophic lateral sclerosis (Lou Gehrig's disease)

The existence of a link between some cases of familial amyotrophic lateral sclerosis (FALS) and copper-zinc superoxide dismutase (CuZnSOD) has been understood for almost a decade. However, beyond the fact that mutations in CuZnSOD cause FALS by a toxic gain of function, the mechanism whereby specific mutations in the protein structure result in development of the disease has remained almost a complete mystery to date. We have undertaken a critical survey of in vitro characteristics of over 30 of the 90 different CuZnSOD mutant proteins that are known to cause FALS in order to determine the differences that exist between mutant and wild-type properties. As-isolated metal content analysis, SOD activity assays, and thermal stability determinations of a significant fraction of the mutants show that the FALS mutant SOD proteins can be classified distinctly into one of two groups. Members of the first group, termed wild-type-like, have physical properties and enzymatic activities that are strikingly similar to those of wild-type CuZnSOD. The second group, however, show aberrant metal content in the as-isolated forms, compromised SOD activities, and unusual DSC thermoscans. All mutations in the members of this second group occur in or near the metal binding sites of the protein and thus they are termed metal binding region mutants. We have also compared the relative rates of self-inactivation caused by reaction of the wild-type protein and several FALS-linked CuZnSOD mutants with hydrogen peroxide, as a measure of relative peroxidative activities. Results and implications of the role of CuZnSOD in FALS are discussed.     

Oxidative DNA cleavage mediated by a new copper (II) terpyridine complex: crystal structure and DNA binding studies

The copper (II) complex [Cu(Itpy)(2)](ClO(4))(2) (1), (Itpy=imidazole terpyridine) has been synthesized and structurally characterized. Crystal structure of the complex shows the complex to be a monomeric copper (II) species with two Itpy ligands coordinated to the metal ion to give a six coordinate complex. The complex has a distorted octahedral geometry with axial elongation. Variable temperature crystal structure data shows dynamic nature of the Jahn-Teller distortion. The complex is an avid DNA binder with a binding constant of 4.26+/-0.20x10(3)M(-1). Observed changes in the viscosity and circular dichroic spectrum of calf thymus DNA solution in the presence of complex 1 suggests intercalative binding of complex 1 to DNA. The complex cleaves supercoiled pBR322 DNA oxidatively in the presence of hydrogen peroxide.     

A structure-based mechanism for copper-zinc superoxide dismutase

A reaction cycle is proposed for the mechanism of copper-zinc superoxide dismutase (CuZnSOD) that involves inner sphere electron transfer from superoxide to Cu(II) in one portion of the cycle and outer sphere electron transfer from Cu(I) to superoxide in the other portion of the cycle. This mechanism is based on three yeast CuZnSOD structures determined by X-ray crystallography together with many other observations. The new structures reported here are (1) wild type under 15 atm of oxygen pressure, (2) wild type in the presence of azide, and (3) the His48Cys mutant. Final R-values for the three structures are respectively 20.0%, 17.3%, and 20.9%. Comparison of these three new structures to the wild-type yeast Cu(I)ZnSOD model, which has a broken imidazolate bridge, reveals the following: (i) The protein backbones (the "SOD rack") remain essentially unchanged. (ii) A pressure of 15 atm of oxygen causes a displacement of the copper ion 0.37 A from its Cu(I) position in the trigonal plane formed by His46, His48, and His120. The displacement is perpendicular to this plane and toward the NE2 atom of His63 and is accompanied by elongated copper electron density in the direction of the displacement suggestive of two copper positions in the crystal. The copper geometry remains three coordinate, but the His48-Cu bond distance increases by 0.18 A. (iii) Azide binding also causes a displacement of the copper toward His63 such that it moves 1.28 A from the wild-type Cu(I) position, but unlike the effect of 15 atm of oxygen, there is no two-state character. The geometry becomes five-coordinate square pyramidal, and the His63 imidazolate bridge re-forms. The His48-Cu distance increases by 0.70 A, suggesting that His48 becomes an axial ligand. (iv) The His63 imidazole ring tilts upon 15 atm of oxygen treatment and azide binding. Its NE2 atom moves toward the trigonal plane by 0.28 and 0.66 A, respectively, in these structures. (v) The replacement of His48 by Cys, which does not bind copper, results in a five-coordinate square pyramidal, bridge-intact copper geometry with a novel chloride ligand. Combining results from these and other CuZnSOD crystal structures, we offer the outlines of a structure-based cyclic mechanism.     

Structure of the Rhodobacter sphaeroides light-harvesting 1 beta subunit in detergent micelles.

The light harvesting 1 antenna (LH1) complex from Rhodobacter sphaeroides funnels excitation energy to the photosynthetic reaction center. Our ultimate goal is to build up the structure of LH1 from structures of its individual subunits, much as the antenna can self-assemble from its components in membrane-mimicking detergent micelles. The beta subunit adopts a nativelike conformation in Zwittergent 3:12 micelles as demonstrated by its ability to take the first step of assembly, binding BChl a. Multidimensional NMR spectroscopy shows that the beta subunit folds as a helix((L12-S25))-hinge((G26-W28))-helix((L29-W44)) structure with the helical regions for the 10 lowest-energy structures having backbone rmsds of 0.26 and 0.24 A, respectively. Mn(2+) relaxation data and the protein-detergent NOE pattern show the C-terminal helix embedded in the micelle and the N-terminal helix lying along the detergent micelle surface with a 60 degrees angle between their long axes. (15)N relaxation data for residues L12-W44 are typical of a well-ordered protein with a correlation time of 8.25 +/- 2.1 ns. The presence of the hinge region placing the N-terminal helix along the membrane surface may be the structural feature responsible for the functional differences observed between the LH1 and LH2 beta subunits.     

Copper(2+) binding to the surface residue cysteine 111 of His46Arg human copper-zinc superoxide dismutase, a familial amyotrophic lateral sclerosis mutant

Mutations in copper-zinc superoxide dismutase (CuZnSOD) cause 25% of familial amyotrophic lateral sclerosis (FALS) cases. This paper examines one such mutant, H46R, which has no superoxide dismutase activity yet presumably retains the gain-of-function activity that leads to disease. We demonstrate that Cu(2+) does not bind to the copper-specific catalytic site of H46R CuZnSOD and that Cu(2+) competes with other metals for the zinc binding site. Most importantly, Cu(2+) was found to bind strongly to a surface residue near the dimer interface of H46R CuZnSOD. Cysteine was identified as the new binding site on the basis of multiple criteria including UV-vis spectroscopy, RR spectroscopy, and chemical derivatization. Cysteine 111 was pinpointed as the position of the reactive ligand by tryptic digestion of the modified protein and by mutational analysis. This solvent-exposed residue may play a role in the toxicity of this and other FALS CuZnSOD mutations. Furthermore, we propose that the two cysteine 111 residues, found on opposing subunits of the same dimeric enzyme, may provide a docking location for initial metal insertion during biosynthesis of wild-type CuZnSOD in vivo.     

Structure of the yeast vacuolar ATPase

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.     

X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery

Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 A crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated.     

In vivo peroxidative activity of FALS-mutant human CuZnSODs expressed in yeast.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to loss of motor neurons. We previously characterized the enhanced peroxidative activity of the human familial ALS (FALS) mutants of copper-zinc superoxide dismutase (CuZnSOD) A4V and G93A in vitro. Here, a similar activity is demonstrated for human FALS CuZnSOD mutants in an in vivo model system, the yeast Saccharomyces cerevisiae. Spin trap adducts of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) have been measured by electron paramagnetic resonance (EPR) in yeast expressing mutant (A4V, L38V, G93A, and G93C) and wild type CuZnSOD upon addition of hydrogen peroxide to the culture. The trapped radical is a hydroxyethyl adduct of POBN, identified by spectral parameters. Mutant CuZnSODs produced greater concentrations of the trapped adduct compared to the wild type enzyme. This observation provides evidence for an oxidative radical mechanism, whereby the mutants of CuZnSOD catalyze the formation of reactive oxygen species that may be related to the development or progression of FALS. This study also presents an in vivo model system to study free radical production in FALS-associated CuZnSOD mutations.     

An oxidized tryptophan facilitates copper binding in Methylococcus capsulatus-secreted protein MopE

Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35 angstroms of MopE* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE*.     

Crystal structure of the archaeal A1Ao ATP synthase subunit B from Methanosarcina mazei Gö1: Implications of nucleotide-binding differences in the major A1Ao subunits A and B

The A1Ao ATP synthase from archaea represents a class of chimeric ATPases/synthases, whose function and general structural design share characteristics both with vacuolar V1Vo ATPases and with F1Fo ATP synthases. The primary sequences of the two large polypeptides A and B, from the catalytic part, are closely related to the eukaryotic V1Vo ATPases. The chimeric nature of the A1Ao ATP synthase from the archaeon Methanosarcina mazei G?1 was investigated in terms of nucleotide interaction. Here, we demonstrate the ability of the overexpressed A and B subunits to bind ADP and ATP by photoaffinity labeling. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to map the peptide of subunit B involved in nucleotide interaction. Nucleotide affinities in both subunits were determined by fluorescence correlation spectroscopy, indicating a weaker binding of nucleotide analogues to subunit B than to A. In addition, the nucleotide-free crystal structure of subunit B is presented at 1.5 A resolution, providing the first view of the so-called non-catalytic subunit of the A1Ao ATP synthase. Superposition of the A-ATP synthase non-catalytic B subunit and the F-ATP synthase non-catalytic alpha subunit provides new insights into the similarities and differences of these nucleotide-binding ATPase subunits in particular, and into nucleotide binding in general. The arrangement of subunit B within the intact A1Ao ATP synthase is presented.     

Resonance raman studies of heme structural differences in subunits of deoxy hemoglobin

Low frequency resonance Raman (RR) spectra are reported for deoxy hemoglobin (Hb), its isolated subunits, its analogue bearing methine-deuterated hemes in all four subunits (Hb-d(4)), and the hybrids bearing the deuterated heme in only one type of subunit, which are [alpha(d4)beta(h4)](2) and [alpha(h4)beta(d4)](2). Analyzed collectively, the spectra reveal subunit-specific modes that conclusively document subtle differences in structure for the heme prosthetic groups in the two types of subunits within the intact tetramer. Not surprisingly, the most significant spectral differences are observed in the gamma(7) mode that has a major contribution from out of plane bending of the methine carbons, a distortion that is believed to relieve strain in the high-spin heme prosthetic groups. The results provide convincing evidence for the utility of selectively labeled hemoglobin hybrids in unraveling the separate subunit contributions to the RR spectra of Hb and its various derivatives and for thereby detecting slight structural differences in the subunits.     

Synthesis, crystal structure and nuclease activity of a Schiff base copper(II) complex

A new Schiff base copper(II) complex, Cu(o-VANAHE)(2) (o-VANAHE = 2-(o-vanillinamino)-1-hydroxyethane), has been synthesized and characterized. Single crystal X-ray diffraction results suggest that this complex structure belongs to triclinic crystal system, space group P1 with the following crystallographic parameters: a = 8.819(4) angstroms, b = 10.794(5) angstroms, c = 11.350(5) angstroms, alpha = 70.262(6) degrees, beta = 70.816(6) degrees, gamma = 78.360(6) degrees, V = 955.4(7) angstroms3, Z = 2, D(c) = 1.571 Mg x m(-3), and the final R1 = 0.0393, wR2 = 0.0994 for the observed reflections 2620(I > 2sigma(I)). The molecular geometry is almost coplanar. Viscosity, fluorescence spectroscopy and cyclic voltammetry have been conducted to assess their interaction between this complex and DNA. Results showed that the copper(II) complex can increase DNA's relative viscosity and quench the fluorescence intensity of EB bound to DNA. The adding of DNA to the solution of Cu(o-VANAHE)2 causes a slight decrease in the voltammetric current, as well as a slight shift in the E(1/2) to less negative potential. The interaction between the complex and DNA has also been investigated by submarine gel electrophoresis, interestingly, we found that the copper(II) complex can cleave circular plasmid pBR322 DNA to nicked and linear forms.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.     

Synthesis and physic-chemical properties of a copper(II) complex with 2-(2-pyridyl)iminotetrahydro-1,3-thiazine hydrochloride-water (1/2) (PyTzHCl.2H2O). Crystal structure of PyTz and [[CuCl(PyTz)]2(mu-Cl)2

The synthesis of 2-(2-pyridyl)iminotetrahydro-1,3-thiazine (PyTz) has been carried out, as well as the determination of its X-ray crystal structure, together with the coordination behaviour and equilibra study of PyTzHCl.2H2O with copper(II) in aqueous solution at 298 K and 0.1 M ionic strength in NaClO4. The formation constants are determined and discussed in terms of the characteristics of the ligand. The compound Di-mu-chloro-bis[chloro[2-(2-pyrydil-kappaN)amino-5,6-dihydro-4H-1,3-thiazine-kappaN]copper] has been isolated and its crystal and molecular structure determined by X-ray analysis. The structure consists of dimeric molecules [Cu2Cl4L2], in which copper ions are bridged by two chloro ligands. The geometry about each copper approximates to a distorted square pyramid with the bridging ligands occupying apical and equatorial sites of each copper ion, while the PyTz ligand and the remaining chloride ion are located in an equatorial plane. The compound was also characterized through elemental analysis, magnetic susceptibility, electron paramagnetic resonance, and electronic and infrared spectroscopies.     

Structural basis of heteromeric smad protein assembly in TGF-beta signaling

The formation of protein complexes between phosphorylated R-Smads and Smad4 is a central event in the TGF-beta signaling pathway. We have determined the crystal structure of two R-Smad/Smad4 complexes, Smad3/Smad4 to 2.5 angstroms, and Smad2/Smad4 to 2.7 angstroms. Both complexes are heterotrimers, comprising two phosphorylated R-Smad subunits and one Smad4 subunit, a finding that was corroborated by isothermal titration calorimetry and mutational studies. Preferential formation of the R-Smad/Smad4 heterotrimer over the R-Smad homotrimer is largely enthalpy driven, contributed by the unique presence of strong electrostatic interactions within the heterotrimeric interfaces. The study supports a common mechanism of Smad protein assembly in TGF-beta superfamily signaling.     

Fluoride inhibition of enolase: crystal structure and thermodynamics

Enolase is a dimeric metal-activated metalloenzyme which uses two magnesium ions per subunit: the strongly bound conformational ion and the catalytic ion that binds to the enzyme-substrate complex inducing catalysis. The crystal structure of the human neuronal enolase-Mg2F2P(i) complex (enolase fluoride/phosphate inhibitory complex, EFPIC) determined at 1.36 A resolution shows that the combination of anions effectively mimics an intermediate state in catalysis. The phosphate ion binds in the same site as the phosphate group of the substrate/product, 2-phospho-D-glycerate/phosphoenolpyruvate, and induces binding of the catalytic Mg2+ ion. One fluoride ion bridges the structural and catalytic magnesium ions while the other interacts with the structural magnesium ion and the ammonio groups of Lys 342 and Lys 393. These fluoride ion positions correspond closely to the positions of the oxygen atoms of the substrate's carboxylate moiety. To relate structural changes resulting from fluoride, phosphate, and magnesium ions binding to those that are induced by phosphate and magnesium ions alone, we also determined the structure of the human neuronal enolase-Mg2P(i) complex (enolase phosphate inhibitory complex, EPIC) at 1.92 A resolution. It shows the closed conformation in one subunit and a mixture of open and semiclosed conformations in the other. The EPFIC dimer is essentially symmetric while the EPIC dimer is asymmetric. Isothermal titration calorimetry data confirmed binding of four fluoride ions per dimer and yielded Kb values of 7.5 x 10(5) +/- 1.3 x 10(5), 1.2 x 10(5) +/- 0.2 x 10(5), 8.6 x 10(4) +/- 1.6 x 10(4), and 1.6 x 10(4) +/- 0.7 x 10(4) M(-1). The different binding constants indicate negative cooperativity between the subunits; the asymmetry of EPIC supports such an interpretation.     

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 ? resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ～392 residues and two L subunits of ～150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ～100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 ? away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.