Preliminary crystallographic analysis of a proteolytically modified form of E. coli single stranded DNA binding protein

A proteolytically modified form of the Escherichia coli single-stranded DNA-Binding protein (SSB) has been crystallized from 15% saturated sodium citrate. Crystals as large as 1.0 mm x 0.3 mm x 0.2 mm were obtained and these diffract beyond 3A resolution. X-ray photographic analysis demonstrated a rhombohedral unit cell of space group R3 with an equivalent triple centered hexagonal unit cell having dimensions of a = b = 62.9A and c = 264.3A. These crystals were judged to be adequate for a three dimensional structure determination.     

Preliminary X-ray crystallographic analysis of canine parvovirus crystals

The first diffraction pattern of a crystalline single-stranded DNA virus has been obtained. Canine parvovirus was crystallized in a monoclinic P21 unit cell with a = 264.4 A, b = 350.3 A, c = 267.8 A and beta = 90.86 degrees (1 A = 0.1 nm). The diffraction pattern extends to at least 2.8 A resolution. Packing of the particles suggests that they have a diameter around 257 A, in excellent agreement with the reported molecular weight of 5.5 x 10(6).     

Purification and biochemical characterization of the complete structure of a proteolytically modified beta-2-microglobulin with biological activity

A modified form of beta-2-microglobulin (beta-2-m) has previously been described to be present in serum from patients suffering from autoimmune diseases, acquired immune deficiency syndrome and small-cell lung cancer [Plesner, T. and Wiik, A. (1979) Scand. J. Immunol. 9, 247-254; Bhalla et al. (1985) Clin. Chem. 31, 1411-1412; Nissen et al. (1984) Clin. Chim. Acta 141, 41-50]. In the present study we describe the purification and characterization of this modified human serum beta-2-m from patients with small-cell lung cancer. Purified urinary beta-2-m was added to the serum samples incubated at 20 degrees C for five days to obtain a higher yield of modified beta-2-m (m-beta-2-m). m-beta-2-m was then purified from serum by gel filtration followed by chromatofocusing of the fractions containing beta-2-m. m-beta-2-m was found to have an apparent molecular mass of 15 kDa and a pI of 5.3 when analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and analytical isoelectric focusing respectively. Amino acid analysis of m-beta-2-m revealed that the protein is missing one lysine residue compared to the composition deduced from the cDNA sequence of beta-2-m. Amino acid sequence analysis showed that m-beta-2-m consists of two polypeptide chains produced by a proteolytic cleavage of beta-2-m in the disulphide loop. After reduction and alkylation of m-beta-2-m the two chains were separated by reverse-phase high-pressure liquid chromatography. By amino acid sequencing, amino acid residues 1-56 and 59-99 were identified in the A and B chains respectively. By comparison of the amino acid composition of m-beta-2-m with the known sequence of beta-2-m it was possible to deduce the existence of a Ser-57 in the A chain. Thus proteolytic cleavage of beta-2-m in the intrachain disulphide loop releases the amino acid Lys-58, which results in a modified form of beta-2-m with a molecular mass of 11,620 Da as determined by amino acid analysis.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

X-ray crystallographic and chromatographic characterization of the crystals of Ca2+-calmodulin complexed with bee venom melittin

Crystals of calmodulin complexed with both Ca2+ and melittin, a peptide from bee venom, have been grown from 2-methyl-2,4-pentanediol solution by using the hanging drop method of vapour diffusion. The crystals belong to space group P2(1)2(1)2(1) with a = 97.3(9) A, b = 56.5(0) A, c = 33.4(9) A and Z = 4. Analyses of the dissolved crystals by high performance liquid chromatography show that the crystals contain a 1:1 complex of calmodulin and melittin. An asymmetric unit contains one such complex and the solvent content of the crystals is 47.5% (v/v).     

Determination of the protein content of crystals formed by Mastigocladus laminosus C-phycocyanin, Chroomonas spec. phycocyanin-645 and modified human fibrinogen using an improved Ficoll density gradient method

We demonstrated for several protein crystals of known protein content that the simple Ficoll density gradient method for crystal density determination as described by Westbrook (1976) often leads to quite erroneous results. In particular, the apparent density of loosely packed crystals can show a tremendous change within the first minutes of measurement. In order to derive the correct protein content the apparent crystal density must be followed as a function of time and has to be extrapolated back to the time of insertion of the crystal into the gradient. The packing densities of four novel protein crystals, formed by Mastigocladus laminosus C-phycocyanin, Chroomonas spec. phycocyanin-645 (two forms), and modified human fibrinogen have been determined and that of proteinase II of Crotalus adamanteus has been corrected. The C-phycocyanin crystals were found to contain (in contrast to earlier results reported by others) only one (alpha beta)-monomer, the phycocyanin-645 crystals two and three (alpha alpha' beta 2)-monomers, respectively, and the fibrinogen crystals one fibrinogen molecule per asymmetric unit.     

X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.     

Preliminary X-ray crystallographic analysis of a modified basic fibroblast growth factor

Human basic fibroblast growth factor (hbFGF) has been modified, with Ala3 and Ser5 substituted by glutamic acid, and the purified recombinant protein has been crystallized. The crystals are triclinic (space group P1) with unit cell parameters a = 31.0 A, b = 33.6 A, c = 34.7 A, alpha = 88 degrees, beta = 85 degrees, gamma = 76 degrees, and they diffract to at least 2 A.     

X-ray diffraction characterization of the dense phases formed by nucleosome core particles

Multiple dense phases of nucleosome core particles (NCPs) were formed in controlled ionic conditions (15-160 mM monovalent salt, no divalent ions), under osmotic pressures ranging from 4.7 x 10(5) to 2.35 x 10(6) Pa. We present here the x-ray diffraction analysis of these phases. In the lamello-columnar phase obtained at low salt concentration (<25 mM), NCPs stack into columns that align to form bilayers, kept separated from one another by a layer of solvent. NCPs form a monoclinic lattice in the plane of the bilayer. For high salt concentration (>50 mM), NCPs order into either a two-dimensional columnar hexagonal phase or into three-dimensional orthorhombic (quasi-hexagonal) crystals. The lamellar and hexagonal (or quasi-hexagonal) organizations coexist in the intermediate salt range; their demixing requires a long time. For an applied pressure P = 4.7 10(5) Pa, the calculated NCPs concentration ranges from approximately 280 to 320 mg/ml in the lamello-columnar phase to 495 to 585 mg/ml in the three-dimensional orthorhombic phase. These concentrations cover the concentration of the living cell.     

Biochemical characterization and preliminary X-ray crystallographic study of the domains of human ZBP1 bound to left-handed Z-DNA

ZBP1 is involved in host responses against cellular stresses, including tumorigenesis and viral infection. Structurally, it harbors two copies of the Zalpha domain containing the Zalpha motif, at its N terminus. Here, we attempted to characterize the Z-DNA binding activities of two Zalpha domains in the human ZBP1, hZalpha(ZBP1) and hZbeta(ZBP1), using circular dichroism (CD). Our results indicated that both hZalpha(ZBP1) and hZbeta(ZBP1) are viable Z-DNA binders, and their binding activities are comparable to those of previously-established Zalpha domains. Additionally, we crystallized hZbeta(ZBP1) in a complex with Z-DNA, d(TCGCGCG)2. The crystal diffracted to 1.45 angstroms, and belongs to the P2(1)2(1)2(1) space group, with the unit-cell parameters: a = 29.53 angstroms, b = 58.25 angstroms, and c = 88.61 angstroms. The delineation of this structure will provide insight into the manner in which diverse Zalpha motifs recognize Z-DNA.     

Crystallization and preliminary X-ray crystallographic analysis of human Geminin coiled-coil domain

Initially discovered in Xenopus laevis, Geminin is a DNA replication initiation inhibitor found in higher eukaryotes. The coiled-coil domain of Human Geminin (termed GemH-37) has been crystallized by the vapor-diffusion sitting-drop method. A complete 1.74 A data set has been collected on a single orthorhombic crystal with unit cell parameters a = 25.25, b = 44.35, c = 68.58 A. Successful molecular replacement shows that GemH-37 is a dimeric parallel coiled-coil. Structural analysis is now in progress.     

Crystallization and preliminary X-ray crystallographic studies of human placental annexin IV

Human placental annexin IV, a member of the annexin family of calcium and phospholipid-binding proteins, has been crystallized by the vapour diffusion method in the presence of calcium, using polyethylene glycol 8000. The crystals are orthorhombic, space C222(1), cell dimensions a = 105.4 A, b = 115.7 A, c = 80.7 A and diffract to at least 2.5 A resolution on a synchrotron source.     

X-ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation

Fumarase catalyzes the reversible conversion of fumarate to S- malate during the operation of the ubiquitous Kreb's cycle. Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme. We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site. Offspring homozygous for the missense mutation, a G-955-C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid. To more fully understand the implications of this mutation, a single-step site-directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli. Subsequent kinetic and X-ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.     

An X-ray crystallographic investigation of exchange of localized sulfate ions in crystals of α-chymotrypsin

Localized sulfate ions in the crystal structure of α-chymotrypsin have been exchanged with selenate ions. The exchange was studied using difference X-ray crystallographic methods at 2.8 Å resolution at three (SeO₄^2?:(SO₄^2?) ion ratios (5:1, 20:1, 100:1) and crystals grown from ammonium selenate. All the localized (SO₄^2? ions are exchangeable and they apparently solvate the enzyme molecule with a range of binding constants. One pair of ions is extremely facile toward the exchange. The latter occur internally at high occupancy in the interface region between the two independent molecules of the asymmetric unit and participate in a hydrogen bonding scheme that appears to be an important interaction in maintaining the dimeric structure of the crystal. These ions form hydrogen bonding bridges in a reciprocating manner between the independent molecules of the asymmetric unit of the crystal which involve the side chain of catalytically crucial Ser195 of one molecule and the phenolic hydroxyl of Tyr′146 of the other molecule; in addition, each (SeO₄^2? ion also appears to hydrogen bond with the amino group of its respective Gly193. The remainder of the (SeO₄^2? ions are at lower occupancies and not nearly as definite. These are located around the surface of the enzyme where they generally interact with a positive charge and/or enter into hydrogen bonding with the enzyme. In general, the (SO₄^2? ions display relatively poor local 2-fold symmetry, probably because of their weak interaction with the enzyme.     

Preliminary X-ray study of crystals of human transferrin

Large crystals of human transferrin have been grown from polyethylene glycol solutions. The crystals are orthorhombic, space group P2₁2₁2₁, with $\text{a = 78}\text{A}\text{?}\text{, b = 94}\text{A}\text{?}\text{and}\text{c = 112}\text{A}\text{?}$. Density measurements indicate that there are four transferrin molecules in the unit cell.