Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman–Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P4_1, with R_work/R_free values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme–inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.     

Crystallization of the bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase domain of the human trifunctional enzyme

Methylenetetrahydrofolate([H₄] folate) dehydrogenase (D) and methenyl[H₄] folate cyclohydrolase (C) coexist as a bifunctional enzyme (DC) or as the amino-terminal domain of a trifunctional enzyme (DCS) where the third activity is 10-formyl[H₄]lfolate synthetase (S). Two crystal forms of the DC domain of the human cytosolic DCS enzyme have been grown from polyethyleneglycol solution. The monoclinic P2₁ crystals diffract to 2.8 Å with a = 72.5 Å, b = 68.5 Å, c = 125.2 Å, and β = 91.8° but were found to be twinned. The orthorhombic P2₁2₁2₁ crystals diffract to 2.5 Å with a = 67.7 Å, b = 135.9 Å, c = 61.6 Å, and contain two molecules per asymmetric unit. Proteins 26:479–480 © 1996 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray studies on the rhizome lectin from stinging nettle and its complex with NN′N″-triacetylchitotriose

Single crystals were grown from affinity-purified stinging nettle lectin and from its complex with the specific trisaccharide NN′N″ -triacetylchitotriose by vapor diffusion at room temperature. The lectin crystallizes in space group P2₁2₁2₁ with unit cell dimensions a = 54.3 (1) Å, b = 62.2 (1) Å, and c = 92.4 (2) Å, and diffracts to 3.0 Å resolution. The asymmetric unit contains three lectin monomers. The crystals of the lectin-trisaccharide complex have space group P2₁2₁2₁ with cell constants a = 37.69 (4) Å, b = 48.97 (6) Å, and c = 57.32 (4) Å. These crystals diffract to at least 2.0 Å resolution and the asymmetric unit contains one lectin monomer. A three-dimensional X-ray structure determination is on its way. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of formylmethanofuran: Tetrahydromethanopterin formyltransferase from Methanopyrus kandleri

Formylmethanofuran:tetrahydromethanopterin formyltransferase from the hyperthermophilic methanogenic Archaeon Methanopyrus kandleri (growth temperature optimum 98°C) was crystallized by vapor diffusion methods. Crystal form M obtained with 2-methyl-2,4-pentanediol as precipitant displayed the space group P2₁ with unit cell parameters of a = 87.0 Å, b = 75.4 Å, c = 104.7 Å, and β = 113.9° and diffracted better than 2 Å resolution. Crystal form P grown from polyethylene glycol 8000 belonged to the space group I4₁22 and had unit cell parameters of 157.5 Å and 242.1 Å. Diffraction data to 1.73 Å were recorded. Crystal form S which was crystallized from (NH₄)₂SO₄in the space group I4₁22 with unit cell parameters of 151.3 Å and 249.5 Å diffracted at least to 2.2 Å resolution. All crystal forms probably have four molecules per asymmetric unit and are suitable for X-ray structure analysis. © 1996 Wiley-Liss, Inc.     

Mechanistic Studies on Metal-pyrophosphate Hydrolysis. Structure of Tetraammine(chloroaquo)cobait(III) dichloride ([CO(NH3)4ClH20]2+•-2Cl−), an Acid Hydrolysis Product of Co(NH3)4HP2O7 and Co(NH3)4PO4

Abstract

The octahedral complex tetraammine(chloroaquo)cobalt(III) dichloride is shown to be the HCl hydrolysis product of both P¹,^2-bidentate tetraammine(pyrophosphato)cobalt(III) [CO(NH₃)₄HP₂0₇ or CoPP] and bidentate tetraammine(phosphato)cobalt(III) [Co(NH₃)₄P0₄or CoP]. The complex crystallizes in the orthorhombic space group Pna2¹ with cell dimensions α=13.033(2)Å, b=6.710(1) Å, and c=10.318(2)Å; the crystal structure was refined to a final disagreement index of 0.033. The average of the four Co-N distances is 1.944±6Å. The Co-Cl distance is 2.257(2)Å and the Co-O(W) distance is 1.971(4)Å. Both protons of the coordinated water molecule are engaged in strong hydrogen bonds to the two nonbonded chloride counterions with 0(W)-C1 distances of 3.087(6)Å and 3.123(6)Å. Each nonbonded chloride is engaged in seven hydrogen bonding interactions resulting from the high ratio of hydrogen bond donors to acceptors in the CoP structure. Cobalt bisphosphate (CoP₂) is the final enzyme hydrolysis product when CoPP is used as substrate in the yeast inorganic pyrophosphatase reaction. The bridge oxygen atom is the site of initial CoPP cleavage both, for HCl catalyzed hydrolysis as well as for enzyme catalyzed hydrolysis.     

Growth and analysis of crystal forms of toxic shock syndrome toxin 1

Native toxic shock syndrom toxin 1 (TSST-1) purified from Staphylococcus aurius has been crystallized in four different forms. The highest resolution data (2.05 Å) was collected from orthorhombic crystals belonging to the space group C222₁. The unit cell dimension are a = 108.7 Å, b = 177.5 Å, c = 97.6 Å. Rotation function analysis of this from indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 Å. The space group is P2₁ with unit cell parameters of a = 44.4 Å, b = 34.0 Å, c = 55.2 Å, β = 93.0°. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley-Liss, Inc.     

Characterization of two crystal forms of Clostridium thermocellum endoglucanase CelC

Endoglucanase CelC from Clostridium thermocellum expressed in Escherichia coli has been crystallized in two different crystal forms by the hanging drop method. Crystals of form I were grown with polyethylene glycol as a precipitant. They are orthorhombic, space group P2₁2₁2₁, with cell dimensions a =51.4 Å, b =84.3 Å, and c =87.5 Å. Crystals of form II, obtained in ammonium sulfate solutions, belong to the tetragonal space group P4₁2₁2 (or P4₃2₁2) with cell dimensions of a = b = 130.7 Å and c = 69.6 Å. Diffraction data to 2.8 Å resolution were observed for both crystal forms with a rotating anode generator. Preliminary oscillation images of the orthorhombic form I crystals using a synchrotron radiation source show diffraction to 2.2 Å resolution, indicating that these crystals are suitable for high resolution crystallographic analysis. © 1994 Wiley-Liss, Inc.     

Expression and crystallization of the yeast Hsp82 chaperone,and preliminary x-ray diffraction studies of the amino-terminal domain

Expression of the Saccharomyces cerevisiae Hsp82 chaperone in a pep4-3- and hsc82-deficient strain of S. cerevisiae yielded over 25% of the total cell protein as intact Hsp82. Similarly, the amino-terminal domain (residues 1–220) of Hsp82 was expressed to 18% of the total cell protein. Crystals of the intact Hsp82 were readily obtained. The crystals were very fragile, suggesting a high solvent content, and diffracted to approximately 8 Å. Tetragonal bipyrimidal crystals of the amino-terminal domain of Hsp82 were readily obtained under a variety of different conditions. The crystals have primitive tetragonal space group (P422, P4₁22, or its enantiomorph P4₃22) with unit cell dimensions of a = 75.1 Å and c = 111.3 Å, contain 60% by volume solvent, and diffract to 2.5 Å resolution. Addition of 25% glycerol to the mother liquor gave rise to large rod-shaped crystals. The crystals diffract to 2.8 Å resolution, have an orthorhombic space group (P222₁, P2₁2₁2, or P2₁2₁2₁) with cell dimensions of a = 45.2 Å, b = 115.4 Å, and c = 116.9 Å, and a solvent content of 58% by volume. © 1996 Wiley-Liss, Inc.     

Crystals of an antibody FV fragment against an integral membrane protein diffracting to 1.28 Å resolution

The F_v fragment of a monoclonal antibody, 7E2 (IgG₁, κ, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for highresolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 51.51 Å, b = 56.15 Å, c = 99.86 Å (1 Å = 0.1 nm) and contain one F _v fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 Å resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics. © 1995 Wiley-Liss, Inc.     

Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 Å, b = 128.9 Å, c = 78.3 Å, β = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P2₁2₁2₁, with a = 79.8 Å, b = 115.2 Å, c = 73.0 Å) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form. © 1994 John Wiley & Sons, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Pseudomonas fluorescens

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm³ within a month. The crystals belong to the trigonal space group P3₁ (or P3₂), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM ) of 2.53 ų/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic analysis of the N-terminal actin binding domain of human fimbrin

We have crystallized the N-terminal actin binding domain (ABD1) of human fimbrin, a representative member of the largest class of actin crosslinking proteins. Diffraction from these crystals is consistent with the orthorhombic space group P2₁2₁2₁ (a = 50.03 Å, b = 61.24 Å, c = 102.30 Å). These crystals contain one molecule in the asymmetric unit and diffract to at least 1.9 Å resolution. The crystal structure of ABD1 will be the first structure of an actin crosslinking domain. Proteins 28:452–453, 1997. © 1997 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of phospholipid transfer protein from maize seedlings

Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (V_m) of 2.36 Å ³/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuK_α and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.     

Expression,purification, and crystallization of restriction endonuclease PvuII with DNA containing its recognition site

We have overexpressed the type II restriction endonuclease PvuII (R.PvuII) in E. coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site. The cocrystals are orthorhombic space group P2₁2₁2₁ with cell constants a = 95.8 Å, b = 86.3 Å, c = 48.5 Å, and diffract X-rays to at least 2.7 Å. There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit. © 1994 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici

The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2₁, with unit cell dimensions a= 102.4 Å b= 116.5 Å c= 115.8 Å and β = 103.5 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 Å₃/Da. The crystals diffract to at least 2.3 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. © 1997 Wiley-Liss, Inc.     

Crystallization,characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22°C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 × 0.3 × 0.7 mm diffract at room temperature to at least 3.5 Å using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitablefor high resolution studies. The space group is P4₁2₁2 (or its enantiomorph P4₃2₁2), with unit cell dimensions a = b = 291.6 Å and c = 189.4 Å. Density packing considerations areconsistent with the presence of 4-6 monomers (M_r of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme. © 1995 Wiley-Liss, Inc.     

Conformation of the cyclic tetrapeptide dihydrochlamydocin. Iabu-L-Phe D-Pro-LX, and experimental values for 3 leads to 1 intramolecular hydrogen bonds by X-ray diffraction.

Chlamydocin, Iabu-L -Phe-D -Pro-L X, is a naturally occurring cyclic tetrapeptide that exhibits high cytostatic activity. The conformation of the peptide ring, as well as the stereo configuration in the vicinity of the epoxide ring, have been established by a single-crystal X-ray study of dihydrochlamydocin: C₂₈H₄₀N₄O₆·H₂O. It crystallizes in the monoclinic space group P2₁ with a = 12.616(6) Å, b = 12.355(6) Å, c = 9.442(5) Å, and β = 99.5(1)°. The structure was solved by the symbolic addition procedure for phase determination followed by the tangent formula method of phase refinement. This structure represents the first cyclic tetrapeptide in which all four peptide units have been found in the trans conformation; however, each peptide unit is significantly nonplanar with ω angles deviating by 14–24° from the ideal value of 180°. This molecule contains two intramolecular 3 → 1 hydrogen bonds and experimentally determined parameters for these seven-membered turns are presented.     

Three-dimensional structure of actinoxanthin. III. A 4-Å resolution

The protein actinoxanthin (molecular weight 10,300) crystallizes in space group P2₁2₁2₁, with cell dimensions a = 30.9 Å, b = 48.8 Å, c = 64.1 Å, and z = 4. The three-dimensional structure of actinoxanthin at 4-Å resolution was determined by x-ray methods on the basis of experimental data from the native protein and five isomorphous derivatives. At the stage of solving the phase problem, the heavy atoms in the derivatives were located using direct methods. The actinoxanthin molecule can be described as an oblate ellipsoid with approximate dimensions 20 × 30 × 40 Å and consists of two different sizes of folded units separated by a well-defined cleft. The larger unit, including the N- and C-terminals of the protein chain, is characterized by a significant content of β-sheet structure. The smaller unit, containing two deca- and hexapeptide cycles closed by disulfide bonds, has a mainly irregular structure.