An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Ppseudomonas putida mt-2.

BACKGROUND: Catechol dioxygenases catalyze the ring cleavage of catechol and its derivatives in either an intradiol or extradiol manner. These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively. The lack of an MPC structure has hampered the understanding of the general mechanism of extradiol dioxygenases. RESULTS: The three-dimensional structure of MPC has been determined at 2.8 A resolution by the multiple isomorphous replacement method. The enzyme is a homotetramer with each subunit folded into two similar domains. The structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2-dioxygenase, although there is low amino acid sequence identity between these enzymes. The active-site structure reveals a distorted tetrahedral Fe(II) site with three endogenous ligands (His153, His214 and Glu265), and an additional molecule that is most probably acetone. CONCLUSIONS: The present structure of MPC, combined with those of two 2,3-dihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the active site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The results suggest that extradiol dioxygenases employ a common mechanism to recognize the catechol ring moiety of various substrates and to activate dioxygen. One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.

Crystallization of the complexes between M315 idiotope and its monoclonal anti-idiotopic antibody Fab fragment.

The Fab fragment of a monoclonal anti-idiotopic antibody against M315 has been isolated and its complexes with Fv and Fab' fragment of M315 have been crystallized by using poly(ethylene glycol) 6000 or ammonium sulfate. X-ray diffraction photographs showed that the crystal of the complex with Fv diffracts better than that with Fab'. The Fv-complexed crystal was shown to be tetragonal I4, with cell dimensions a = 152 A and c = 69 A, and to contain one complex molecule of about 75,000 molecular weight in the crystallographic asymmetric unit.

Structural changes of active site cleft and different saccharide binding modes in human lysozyme co-crystallized with hexa-N-acetyl-chitohexaose at pH 4.0.

Human lysozyme was co-crystallized with hexa-N-acetyl-chitohexaose, (GlcNAc)6, at pH 4.0 and 4.0 degrees C in a new orthorhombic form, where two protein molecules, MOL1 and MOL2, were contained in an asymmetric unit. The three-dimensional structure was refined to an R-factor of 17.0% at 1.6 A resolution. It was found that (GlcNAc)6 had already been cleaved to (GlcNAc)4 and (GlcNAc)2. In MOL1, (GlcNAc)4 was bound to the A, B, C, and D subsites, and binding sites of (GlcNAc)2 were close to the E and F subsites proposed on the basis of model building by Phillips and his colleagues. In MOL2, only the (GlcNAc)4 moiety could be found in the A, B, C and D subsites. Significant shifts of the backbone atoms were observed in the region of residues 102 to 120, which composed one side of the wall of the active site cleft. Consequently, the active cleft, with respect to the saccharide binding sites A, B and C, is narrower in both protein molecules. The residues 109 to 111 in site D of MOL1 are moved toward saccharide residue D, whereas those of MOL2 are only slightly shifted. In spite of these facts, the saccharide residues in site MOL1 and MOL2 are moved inside of the cleft. The distribution of water molecules and the hydrogen bond network in site D differ between the structures of MOL1 and MOL2. These structural changes in the active site cleft may be responsible for accommodating the substrate and releasing the products of hydrolysis. These results suggest that the three-dimensional structures of MOL1 and MOL2 remain in intermediate states between a transition state and an enzyme/product complex state.

Normal mode refinement: crystallographic refinement of protein dynamic structure. II. Application to human lysozyme.

The dynamic structure of a protein, human lysozyme, is determined by the normal mode refinement of X-ray crystal structure. This method uses the normal modes of both internal and external motions to distinguish the real internal dynamics from the external terms such as lattice disorder, and gives an anisotropic and concerted picture of atomic fluctuations. The refinement is carried out with diffraction data of 5.0 to 1.8 A resolution, which are collected on an imaging plate. The results of the refinement show: (1) Debye-Waller factor consists of two parts, highly anisotropic internal fluctuations and almost isotropic external terms. The former is smaller than the latter by a factor of 0.72 in the scale of B-factor. Therefore, the internal dynamics cannot be recognized directly from the apparent electron density distribution. (2) The internal fluctuations show basically similar features as those predicted by the normal mode analysis, with almost the same amplitude and a similar level of anisotropy. (3) Correlations of fluctuations are detected between two lobes forming the active site cleft, which move simultaneously in opposite directions. This corresponds to the hinge-bending motion of lysozyme.

Crystallization of mutant beta subunit of F1-ATPase from thermophilic Bacillus PS3.

The mutant beta subunit of F1-ATPase from a thermophilic Bacillus strain, PS3, in which tyrosine at position 341 is replaced by leucine (beta Y341L) was expressed in Escherichia coli and crystallized by the vapor-diffusion procedure. Small needle-like crystals were obtained using ammonium sulfate as a precipitant and grown by the stepwise seeding method. The crystals obtained by this procedure diffracted X-rays to about 3 A resolution. The diffraction patterns indicated that the crystals belong to the orthorhombic system and the space group I222 or I2(1)2(1)2(1) with unit-cell dimensions of a = 232 A, b = 66 A, and c = 80 A. It is thought that the asymmetric unit comprises one beta Y341L molecule.

Crystallization and preliminary X-ray diffraction studies of photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans.

Photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans was crystallized by the hanging drop vapor diffusion procedure using ammonium sulfate as a precipitant. The pale-yellow crystals were grown to a size of 0.4 mm in length and 0.1 mm in diameter. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 90.7 A and c = 135 A. Assuming that the asymmetric unit contains one molecule, the Vm value is calculated as 2.6 A3/dalton. The crystals are stable towards X-ray exposure and diffract beyond 2.5 A resolution.

Response of dynamic structure to removal of a disulfide bond: normal mode refinement of C77A/C95A mutant of human lysozyme.

In order to investigate the response of dynamic structure to removal of a disulfide bond, the dynamic structure of human lysozyme has been compared to its C77A/C95A mutant. The dynamic structures of the wild type and mutant are determined by normal mode refinement of 1.5-A-resolution X-ray data. The C77A/C95A mutant shows an increase in apparent fluctuations at most residues. However, most of the change originates from an increase in the external fluctuations, reflecting the effect of the mutation on the quality of crystals. The effects of disulfide bond removal on the internal fluctuations are almost exclusively limited to the mutation site at residue 77. No significant change in the correlation of the internal fluctuations is found in either the overall or local dynamics. This indicates that the disulfide bond does not have any substantial role to play in the dynamic structure. A comparison of the wild-type and mutant coordinates suggests that the disulfide bond does not prevent the 2 domains from parting from each other. Instead, the structural changes are characteristic of a cavity-creating mutation, where atoms surrounding the mutation site move cooperatively toward the space created by the smaller alanine side chain. Although this produces tighter packing, more than half of the cavity volume remains unoccupied, thus destabilizing the native state.

Specific arrangement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase and the other flavin-dependent reductases.

The structure of NADH-cytochrome b5 reductase from pig liver microsomes has been refined to a crystallographic R factor of 0.223 at 2.4 A resolution. A structural comparison between the flavin-binding beta barrel domain of NADH-cytochrome b5 reductase and those of the other flavin-dependent reductases, ferredoxin-NADP+ reductase, phthalate dioxygenase reductase and nitrate reductase, indicated that the overall barrel foldings are similar to each other and that the specific arrangement of three amino acid residues (Arg, Tyr and Ser/Thr) is usually necessary for flavin-binding. These conserved residues overlap each other in their three-dimensional structures and stabilize the flavin-binding site in the four flavin-dependent reductases.

PDI and glutathione-mediated reduction of the glutathionylated variant of human lysozyme.

A mutant human lysozyme, designated as C77A-a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)-lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h-lysozyme C77A-a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.

Preliminary X-ray crystallographic studies of photosynthetic reaction center from a thermophilic sulfur bacterium, Chromatium tepidum.

A membrane protein complex, photosynthetic reaction center purified from the thermophilic purple sulfur bacterium, Chromatium tepidum has been crystallized from a phosphate buffer containing a detergent, n-octyl-beta-D-glucopyranoside and a precipitant, polyethylene glycol 4000. The crystals diffracted X-rays beyond 3A resolution with synchrotron radiation and are suitable for high-resolution X-ray crystallographic studies. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions of a = 136A, b = 197A, and c = 82A. Assuming that they contain one reaction center complex in the asymmetric unit, VM was calculated to be 4.3 A3/Da, which agrees with the values obtained in the membrane protein complexes.

Crystallization and preliminary X-ray diffraction studies of expressed Pseudomonas putida catechol 2,3-dioxygenase.

Crystals of recombinant Pseudomonas putida catechol 2,3-dioxygenase, metapyrocate-chase, composed of four identical subunits, each with a molecular mass of 35 kDa and one nonheme ferrous iron, have been grown by the vapor diffusion method using sodium citrate as the precipitant. Repeated macroseeding and the addition of ethanol to protein solutions were together effective for obtaining crystals suitable for further crystallographic characterization. The crystals belong to the tetragonal space group P4(2)2(1)2 with unit-cell dimensions of a = b = 266 A, c = 60 A. They diffracted beyond 2.5 A resolution with synchrotron radiation. Assuming that one tetramer (alpha-Fe2+)4 is contained in an asymmetric unit, the crystal volume per unit molecular mass, Vm, is calculated to be 3.8 A3/Da, which corresponds to the solvent content of 67.6%.

Crystal structures of the apo- and holomutant human lysozymes with an introduced Ca2+ binding site.

The three-dimensional structures of apo- and holomutant human lysozymes (D86/92 lysozyme), in which a calcium binding site was designed and created for enhancing molecular stability by replacing both Gln86 and Ala92 with aspartic acids, were refined at 1.8-A resolution by x-ray crystallography. The overall structures and crystallographic thermal factors of all three proteins, the apo-, holo-D86/92, and the wild-type human lysozymes, were essentially identical; these results showed that the introduction of the calcium binding site did not affect either the overall structure or molecular rigidity of the proteins. However, structure analyses of the apo-D86/92 lysozyme revealed that the mutations affected the side chain conformation of residue 86 and hydrogen networks between the protein and the internal solvent molecules. In the structure of the holo-D86/92 lysozyme, seven oxygen ligands formed a slightly distorted pentagonal bipyramid around the calcium ion, indicating that the coordination around the calcium ion was quite similar to that in baboon alpha-lactalbumin. The pentagonal bipyramid coordination could be one of the most widely found and appropriate calcium binding schemes in proteins.

Crystal structure of a mutant human lysozyme with a substituted disulfide bond.

The three-dimensional structure of a mutant human lysozyme, W64CC65A, in which a non-native disulfide bond Cys64--Cys81 is substituted for the Cys65--Cys81 of the wild type protein by replacing Trp64 and Cys65 with Cys and Ala, respectively, was determined by X-ray crystallography and refined to an R-value of 0.181, using 33,187 reflections at 1.87-A resolution. The refined model of the W64CC65A protein consisted of four molecules, which were related by two noncrystallographic twofold axes and a translation vector. Although no specific structural differences could be observed among these four molecules, the overall B-factors of each molecule were quite different. The overall structure of W64CC65A, especially in the alpha-helical domain, was found to be quite similar to that of the wild type protein. Moreover, the side-chain conformation of the newly formed Cys64--Cys81 bond was quite similar to that of the Cys65--Cys81 bond of the wild-type protein. However, in the beta-sheet domain, the main-chain atoms of the loop region from positions 66-75 could not be determined, and significant structural changes due to the formation of the non-native disulfide bond could be observed. From these results, it is clear that the loop region of the mutant protein does not fold with the specific folding as observed in the wild-type protein.

Normal mode refinement: crystallographic refinement of protein dynamic structure applied to human lysozyme.

A new method of dynamic structure refinement of protein x-ray crystallography, normal mode refinement, is developed. In this method the Debye-Waller factor is expanded in terms of the low-frequency normal modes and external normal modes, whose amplitudes and couplings are optimized in the process of crystallographic refinement. By this method, internal and external contributions to the atomic fluctuations can be separated. Also, anisotropic atomic fluctuations and their interatomic correlations can be determined experimentally even with a relatively small number of adjustable parameters. The method is applied to the analysis of experimental data of human lysozyme to reveal its dynamic structure.

The crystal structure of a mutant human lysozyme C77/95A with increased secretion efficiency in yeast.

The three-dimensional structure of a mutant human lysozyme, C77/95A, in which residues Cys77 and Cys95 were replaced by alanine, was determined at 1.8-A resolution by x-ray crystallography. The properties of this mutant protein have been well characterized with respect to its thermal stability and secretion efficiency in a yeast expression system. The overall three-dimensional structure of C77/95A was found to be essentially identical to that of the wild-type human lysozyme, although the coordinates were shifted by more than 0.5 A and the thermal factors of the main-chain atoms were increased in the vicinity of residue 77. The reduction in thermal stability of this mutant has been previously explained by an increase in entropy of the unfolded state. In addition, a packing defect (cavity) produced by the removal of the disulfide bond was detected in the three-dimensional structure of C77/95A. This cavity can also be a reason why the stability of the protein is reduced because the free energy of the folded state could be expected to increase. The increased secretion efficiency cannot be due mainly to the three-dimensional structure, but may possibly be related to some event in the pathway of protein secretion. One of the possibilities might involve molecular flexibilities in the secondary or tertiary structure for lack of one of the disulfide bonds.

Structure of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme.

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme. The CRGDSC-inserted lysozyme was expressed in yeast, purified, and designated as Cys-RGD4. Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inserted lysozyme, RGD4. The Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional structure was determined at 1.6-A resolution by x-ray crystallography. In contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was well defined. The structural analysis revealed that the two inserted Cys residues form a new disulfide bond in Cys-RGD4, as expected, and that the RGD region assumes a type II' beta-turn conformation of Gly-Asp with a hydrogen bond between the C = O of Arg and the H-N of Ser. In addition, it was confirmed that two more hydrogen bonds are present in the RGD region of the Cys-RGD4 lysozyme. These results suggest that the conformation of the RGD-containing region is rigid and stable in the Cys-RGD4 molecule and that the type II' beta-turn structure of RGD is essential for binding to integrins with high affinity.

Crystal structure of NADH-cytochrome b5 reductase from pig liver at 2.4 A resolution.

The three-dimensional structure of NADH-cytochrome b5 reductase from pig liver microsomes has been determined at 2.4 A resolution by X-ray crystallography. The molecular structure reveals two domains, the FAD binding domain and the NADH domain. A large cleft lies between these two domains and contains the binding site for the FAD prosthetic group. The backbone structure of the FAD binding domain has a great similarity to that of ferredoxin-NADP+ reductase [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60-65], in spite of the relatively low sequence homology (about 15%) between the two enzymes. On the other hand, the structure of the NADH domain has several structural differences from that of the NADP+ domain of ferredoxin-NADP+ reductase. The size of the cleft between the two domains is larger in NADH-cytochrome b5 reductase than in ferredoxin-NADP+ reductase, which may be responsible for the observed difference in the nucleotide accessibility in the two enzymes.

Preliminary X-ray diffraction study of a new crystal form of C-1027-AG, the apoprotein of the macromolecular antitumor antibiotic C-1027 from Streptomyces globisporus.

A new crystal form of C-1027-AG, the apoprotein of the macromolecular antitumor antibiotic C-1027 isolated from Streptomyces globisporus was obtained by the vapor-diffusion procedure using lithium sulfate as a precipitant. In the present crystallization, it is noteworthy that large-sized single crystals successfully grew from very small droplets (less than 1.01 micro l). The present crystals belong to the trigonal system, space group P3(1)21 or P3(2)21 with cell dimensions of a = b = 62.6 and c = 54.2 A. Assuming that the asymmetric unit contains one molecule, the V(m) value is calculated as 2.9 A(3) Da(-1). A total of 3654 independent reflections from two native crystals was obtained up to 2.5 A resolution with synchrotron radiation, the merging R factor being 0.097.

Structure of a glutathionylated human lysozyme: a folding intermediate mimic in the formation of a disulfide bond.

The three-dimensional structure of a mutant human lysozyme, C77A-a, in which the residue Cys77 is replaced by alanine, has been refined to an R value of 0.125 using 8230 reflections in the resolution range 10.0-1.8 A. It has been shown that C77A-a, in which the counterpart of Cys77 (Cys95) is modified with glutathione, has been shown to mimic an intermediate in the formation of the disulfide bond Cys77-Cys95 during the folding of human lysozyme [Hayano, Inaka, Otsu, Taniyama, Miki, Matsushima & Kikuchi (1993). FEBS Lett. 328, 203-208]. An earlier structure demonstrates that its overall structure is essentially identical to that of the wild-type protein and served as the starting model. The refined model includes atoms for all protein residues (1-130), 20 glutathione atoms and 113 water atoms. Further refinement shows more clearly the details of the protein, the bound glutathione molecule and solvent structure. However, the main-chain folding and the atomic thermal factors of the loop region from Thr70 to Leu79 were highly affected by the binding of the glutathione molecule, as compared with those of the wild-type protein. The bound glutathione shifted the main-chain atoms from Va174 to Ala77 by more than 6.0 A, and the temperature factors of the atoms in the loop region were quite high (more than 40 A(2)), indicating that the backbone conformation of this region is highly flexible and that the loop region is not folded in the specific conformation observed in the wild-type protein. These results strongly suggest that the loop structure in human lysozyme is folded later than the other regions of the protein in vivo, as observed in in vitro folding. Since the bound glutathione is efficiently and irreversibly dissociated by protein disulfide isomerase, the glutathione molecule may act as a protecting group to prevent the formation of an incorrect disulfide bond in the protein folding process in vivo.