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.

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme.

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.

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.

Crystal structure of DNA photolyase from Anacystis nidulans.

The crystal structure at 1.8 A resolution of 8-HDF type photolyase from A. nidulans shows a backbone structure similar to that of MTHF type E. coli photolyase but reveals a completely different binding site for the light-harvesting cofactor.

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%.

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.

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.

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.

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.

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.

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.

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.

Stabilization and enhanced enzymatic activities of a mutant human lysozyme C77/95A with a cavity space by amino acid substitution.

Amino acid substitutions were examined to increase the stability of the mutant human lysozyme C77/95A by filling the cavity created by this mutation. To modulate the cavity with hydrophobic amino acids or by the formation of a hydrogen bond, five amino acid-substituted mutants, C77AC95L, C77AC95I, C77LC95A, C77IC95A and C77/95S, were designed and constructed based on computer graphics investigations for stabilizing the mutant protein. The values of the melting temperatures, Tm, at pH 3.0 of the two mutant proteins C77LC95A and C77/95S were increased by 2.9 degrees C and 2.3 degrees C, respectively, as compared to that of C77/95A. The C77IC95A and C77AC95L proteins showed almost the same stability as C77/95A. The increase in the stability of the proteins might be explained by the filling of the cavity space around positions 77 and 95 with the side residue of Leu77 in C77LC95A, and by the formation of a hydrogen bond between Ser77 and Ser95 in C77/95S. On the other hand, the substitution with isoleucine at 95 (C77AC95I) decreased the stability. The activities of the five mutant proteins against the synthetic substrate, p-nitrophenyl tetra-N-acetyl-beta-chitopentaoside, were higher than that of the wild-type human lysozyme, while the lytic activities against M. lysodeikticus were decreased in C77LC95A and C77IC95A, and increased in C77AC95L.

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.

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.

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.

Crystallization and preliminary X-ray diffraction studies of C-1027-AG, the apoprotein of the macromolecular antitumor antibiotic C-1027 from Streptomyces globisporus.

C-1027-AG, the apoprotein of the macromolecular antitumor antibiotic C- 1027, isolated from Streptomyces globisporus, was crystallized by the vapor-diffusion procedure using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell dimensions a = 55.1, b = 61.3 and c = 79.1 A. Assuming that the asymmetric unit contains two or three molecules, the V(m) value is calculated as 3.2 or 2.1 A(3) Da(-1), respectively. A total of 7630 independent reflections was obtained up to 2.5 A resolution with synchrotron radiation, the merging R factor being 0.077 for 24 713 measurements.

Structural and functional analyses of the Arg-Gly-Asp sequence introduced into human lysozyme.

To determine the functional conformation of the Arg-Gly-Asp (RGD) sequence, we have constructed mutant proteins by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Val74 and Asn75 of human lysozyme. RGDS-, GRGDSP-, TGRGDSPA-, VTGRGDSPAS-, and AVTGRGDS-PASS-introduced mutant lysozymes were expressed in yeast, purified, and designated as RGD4, -6, -8, -10, and -12, respectively. Using baby hamster kidney cells, RGD8, RGD10, and RGD12 were shown to possess high cell adhesion activity nearly equal to 10% of human vitronectin activity. RGD4 and RGD6 exhibited somewhat lower cell adhesion activity. The activities of these mutant proteins were inhibited by the addition of either GRGDSP peptide or polyclonal antibody against vitronectin receptor, as was the case for the vitronectin activity. The results suggest that the cell adhesion signals are transduced to cells through the interaction with the vitronectin receptor. The three-dimensional structures of RGD4 and RGD8 were determined at 1.8-A resolution by x-ray crystallography. A model of the inserted region in RGD4 could be built in the electron density map, but the positions of the preceding residues, Ala73-Val74, were uncertain. The inserted region in RGD8 did not demonstrate continuous electron densities. The results suggest that these RGD sequence-containing regions are highly flexible and that such flexibility could allow the conformation of the RGD regions to be induced to fit into the binding pocket of the integrin receptor.

Enthalpic destabilization of a mutant human lysozyme lacking a disulfide bridge between cysteine-77 and cysteine-95.

To understand the role of disulfide bridges in protein stability, the thermodynamic changes in the denaturation of two mutant human lysozymes lacking a disulfide bridge between Cys-77 and Cys-95 (C77A and C77/95A) were analyzed using differential scanning calorimetry (DSC). At pH 3.0 and 57 degrees C, the stabilities of both the C77A and C77/95A mutants were decreased about 4.6 kcal.mol-1 in Gibbs free energy change. Under the same conditions, the enthalpy changes (delta H) were 94.8 and 90.8 kcal.mol-1, respectively, which were smaller than that of the wild type (100.8 kcal.mol-1). The destabilization of the mutants was caused by enthalpic factors. Although X-ray crystallography indicated that the mutants preserve the wild-type tertiary structure, removal of the disulfide bridge increased the flexibility of the native state of the mutants. This was indicated both by an increase in the crystallographic thermal factors (B-factors) and by a decrease in the affinity of N-acetylglucosamine trimer [(NAG)3] observed using isothermal titration calorimetry (DTC) due to entropic effects. Thus, the effect of cross-linking on the stability of a protein is not solely explained by the entropy change in denaturation.