Two-dimensional assembly of pentameric rabbit C-reactive proteins on lipid monolayers

The problem of pentamer packing on a two-dimensional plane is of concern not only in physics and mathematics but also in biology. The packing styles of pentamers may either be related to or reflect the physiological or biochemical properties of biological macromolecules. C-reactive protein (CRP), one of the classical members of the petraxin family, was recently two-dimensionally (2D) crystallized by us on lipid monolayers by specific adsorption (Wang, H. W., and Sui, S. F., 1999, J. Struct. Biol. 127, 283-286). Another type of the protein's 2D crystal under the same conditions was obtained in the present work. The new 2D crystal was studied using electron microscopy of negatively stained specimens followed by image processing. A projection map at 2.2-nm resolution was obtained. The previous 2D crystal (PI) and the current 2D crystal (PII) show different pentamer-packing styles. Both of them are closely related to the fivefold symmetry of the molecule itself. The coexistence and the spatial contiguity of the two types of pentamer assembly were observed in a visual field. The fivefold symmetrical macromolecule can form a pentiling pattern on a two-dimensional plane, which has never been reported in biological system before. The possible mechanism of the two-dimensional assembly of pentameric CRP on lipid monolayers is discussed.     

A complementary microscopy analysis of Sticholysin II crystals on lipid films: Atomic force and transmission electron characterizations

A new crystal form of the cytotoxin Sticholysin II (StnII) formed on lipid monolayers of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) has been characterized by transmission electron microscopy (TEM) and by tapping mode atomic force microscopy (AFM) under nearly physiological conditions. Both approaches show the existence of single- and double-layered 2D crystals possessing hexagonal symmetry and unit cell dimensions of a = b =10 nm and gamma = 120 degrees. However, single-layered StnII crystals could only be analysed by TEM and double-layered crystals by AFM. Considering the previously known atomic structure of native StnII and that of a tetrameric assembly, a model is proposed for this new crystal form in which StnII conserves its monomeric state upon interaction with the lipid monolayer. These results are in agreement with the existence of the so called M2 state of the actinoporins.     

Partially folded states of the cytolytic protein sticholysin II

Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature < 35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strength > or = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin.     

Electron crystallographic analysis of two-dimensional streptavidin crystals coordinated to metal-chelated lipid monolayers.

Coordination of individual histidine residues located on a protein surface to metal-chelated lipid monolayers is a potentially general method for crystallizing proteins in two dimensions. It was shown recently by Brewster angle microscopy (BAM) that the model protein streptavidin binds via its surface histidines to Cu-DOIDA lipid monolayers, and aggregates into regularly shaped domains that have the appearance of crystals. We have used electron microscopy to confirm that the domains are indeed crystalline with lattice parameters similar to those of the same protein crystallized beneath biotinylated lipid monolayers. Although BAM demonstrates that the two-dimensional protein crystals grown via metal chelation are distinct from the biotin-bound crystals in both microscopic shape and thermodynamic behavior, the two crystal types show similar density projections and the same plane group symmetry.     

Preliminary X-ray crystallographic studies on alcohol dehydrogenase from Drosophila.

The alcohol dehydrogenase (ADHase) enzyme catalyses the oxidation of alcohols to aldehydes or ketones using NAD+ as a cofactor. Functional ADHase from Drosophila lebanonensis is a dimer, with a monomeric molecular weight of 27,000 and with 254 residues in each polypeptide chain. Crystals of the protein have been grown with and without NAD+. Two crystal forms have been observed. Most crystals are plate-like, 0.05 mm in their shortest dimension and up to 0.4 mm in their longest dimension. These crystals are generally too small to diffract efficiently using conventional X-ray sources, so preliminary studies were carried out using the Synchrotron Radiation Source at the SERC Daresbury Laboratory. Twinning was a severe problem with this crystal form. The second form is grown in the absence of NAD+ but with DL-dithiothreitol present. These crystals grow more evenly and diffract to better than 2 A resolution. They are monoclinic, with cell dimensions, a = 81.24(6) A, b = 55.75(4) A, c = 109.60(7) A and beta = 94.26(9) degrees, space group P2(1). There are two dimers in the asymmetric unit, but at low resolution a rotated cell with one dimer per asymmetric unit can be obtained.     

Pentameric Two-Dimensional Crystallization of Rabbit C-Reactive Protein on Lipid Monolayers

As a member of the pentraxin family, C-reactive protein plays various roles in the nonspecific immunity of animals. Though soluble, C-reactive protein always functions on membranes. In order to study the structure of the membrane-bound protein and the reaction between protein and membranes, two-dimensional (2D) crystallization of rabbit C-reactive protein on lipid monolayers was performed. The 2D crystals composed of pentameric proteins were obtained on lipid monolayers by specific adsorption for the first time. The projection map at 26-A resolution is presented, which exhibits P2 symmetry with lattice parameters a = 158(+/-3) A, b = 92(+/-1) A, and gamma = 107(+/-1) degrees. The current work may give a basis for the further study on the structure of complexes made up of C-reactive protein with its functional binding molecules on membranes.     

Crystallization of thermitase, a thermostable subtilisin, from a sodium formate solution by means of an automated procedure

A new crystal form of native thermitase has been obtained using sodium formate as the precipitating agent and employing an automated crystallization procedure. The crystals have the form of tetragonal bipyramids, the longest dimension being about 0.4 mm. The space group is P4(1)2(1)2 or P4(3)2(1)2, with a = 182 A and b = c = 53.3 A. The crystals diffract beyond 2.5 A.     

Global conformational changes control the reactivity of methane monooxygenase.

We present here X-ray scattering data that yield new structural information on the multicomponent enzyme methane monooxygenase and its components: a hydroxylase dimer, and two copies each of a reductase and regulatory protein B. Upon formation of the enzyme complex, the hydroxylase undergoes a dramatic conformational change that is observed in the scattering data as a fundamental change in shape of the scattering particle such that one dimension is narrowed (by 25% or 24 A) while the longest dimension increases (by 20% or 25 A). These changes also are reflected in a 13% increase in radius of gyration upon complex formation. Both the reductase and protein B are required for inducing the conformational change. We have modeled the scattering data for the complex by systematically modifying the crystal structure of the hydroxylase and using ellipsoids to represent the reductase and protein B components. Our model indicates that protein B plays a role in optimizing the interaction between the active centers of the reductase and hydroxylase components, thus, facilitating electron transfer between them. In addition, the model suggests reasons why the hydroxylase exists as a dimer and that a possible role for the outlying gamma-subunit may be to stabilize the complex through its interaction with the other components. We further show that proteolysis of protein B to form the inactive B' results in a conformational change and B' does not bind to the hydroxylase. The truncation thus could represent a regulatory mechanism for controlling the enzyme activity.     

Structure of form III crystals of bovine pancreatic trypsin inhibitor

The structure of bovine pancreatic trypsin inhibitor has been solved in a new crystal form III. The crystals belong to space group P2(1)2(1)2 with a = 55.2 A, b = 38.2 A, c = 24.05 A. The structure was solved on the basis of co-ordinates of forms I and II of the inhibitor by molecular replacement, and the X-ray data extending to 1.7 A were used in a restrained least-squares refinement. The final R factor was 0.16, and the deviation of bonded distances from ideality was 0.020 A. Root-mean-square discrepancy between C alpha co-ordinates of forms III and I are 0.47 A, whilst between forms II and III the discrepancy is 0.39 A. These deviations are about a factor of 3 larger than the expected experimental errors, showing that true differences exist between the three crystal forms. Two residues (Arg39 and Asp50) were modeled with two positions for their side-chains. The final model includes 73 water molecules and one phosphate group bound to the protein. Sixteen water molecules occupy approximately the same positions in all three crystal forms studied to date, indicating their close association with the protein molecule. Temperature factors also show a high degree of correlation between the three crystal forms.     

Crystallization and preliminary X-ray study of AMP nucleosidase

Adenosine-5'-monophosphate nucleosidase from Escherichia coli has been crystallized in the presence of its strong competitive inhibitor formycin 5'-monophosphate and its allosteric activator adenosine 5'-triphosphate. Crystals are tetragonal bipyramids which grow to 1.2 mm in the longest dimension, are resistant to radiation damage, and diffract to a resolution of 3.5 A. The space group is P4(1)2(1)2 or P4(3)2(1)2, and the unit cell dimensions are a = 120.1 A and c = 243.7 A. The asymmetric unit is estimated to contain four subunits of 52,000 daltons. The crystals appear suitable for single crystal x-ray structure investigation.     

Calcium binding to phospholipid: structural study of calcium glycerophosphate.

To consider possible interaction of the phospholipid membrane with calcium ions, crystal structures of calcium dl-alpha- and beta-glycerophosphates (alpha- and beta-CaGs, respectively) were investigated by X-ray diffraction methods. After many attempts, relatively large single crystals of beta-CaG were prepared from the aqueous solution containing HCl, while crystals of CaHPO4.2H2O were obtained from alpha-CaG solution under the same crystallization conditions. The crystal structure of beta-CaG is orthorhombic with space group Pna2(1) and cell dimensions of a = 8.251(1), b = 13.038(3), c = 25.483 (10) A, V = 2741.5 (13) A3 and Z = 16 [four molecules (A to D) in an asymmetric unit]. Molecules of A to D took, as a whole, similar extended conformations, although A and B were different from C and D in the orientation about a glycerol C-C bond. Four independent beta-glycerophosphates commonly act as two types of bidentate ligands, where one is the coordination to the calcium ion by the glycerol O(1) and phosphate O(22) atoms, and the other by the phosphate O(22) and O(23) atoms, thus forming the calcium coordination of a distorted square plane, respectively. Each of four independent calcium ions forms the same coordination geometry of a distorted pentagonal bipyramid. Infinite double layers consisting of alternate A/B molecules and of alternative C/D ones and sandwiching calcium ions were arranged face-to-face along the b-direction and were piled up in the a-direction, thus forming the stacked bilayer unit with the thickness of d002 = 12.75 A. The elaborate networks of calcium coordinations and hydrogen bondings were formed among the layers and stabilized the crystal structure. Based on the structural parameters of the present beta-CaG crystal, a possible interaction model of phospholipid with calcium ions was proposed.     

Polymorphism in beta-cyclodextrin-benzoic acid inclusion complex: a kinetically controlled crystal growth according to the Ostwald's rule

Crystal form II of the beta-cyclodextrin-benzoic acid (beta-CD-BA) inclusion complex was obtained from the 1.5-year stored aqueous EtOH solution of beta-CD and BA as 2beta-CD.1.5BA.0.7EtOH.21H(2)O in the monoclinic space group C2 with unit cell constants: a=19.413(3), b=24.306(4), c=32.975(1)A, beta=104.41(1) degrees . By contrast, the desired crystal form I in the triclinic space group P1 that ever grew up from the fresh solution as 2beta-CD.2BA.0.7EtOH.20.65H(2)O was not reproducible any more [Aree, T.; Chaichit, N. Carbohydr. Res.2003, 338, 439-446]. In the two crystal forms, beta-CDs are isostructural with a 'round' conformation stabilized by intramolecular O-3(n)cdots, three dots, centeredO-2(n+1) hydrogen bonds. The BA inclusion geometries are similar with a preferred orientation, that is, BAs are situated above the O-4 plane, point their COOH groups to the beta-CD O-6 side, incline 52 degrees with respect to the O-4 plane and are mainly maintained in positions by hydrogen bonding with the surrounding water molecules. beta-CDs form dimers as structural motif of different packing modes: the screw-channel type in form II and the average of intermediate and tetrad types in form I. Polymorphism in the beta-CD-BA inclusion complex is a kinetically controlled crystal growth following the Ostwald's rule: the less stable crystal form I grew up first within one week from the fresh solution, whereas the more stable crystal form II appeared after 1.5-year storage.     

Synthesis, structural, physico-chemical and biological properties of new palladium(II) complexes with 2,6-dimethyl-4-nitropyridine

This paper describes the synthesis and properties of two new palladium(II) complexes with 2,6-dimethyl-4-nitro-pyridine (dmnp): mononuclear [Pd(dmnp)2Cl2] and dinuclear [Pd2(dmnp)2Cl4]. Complexes were characterized on the basis of chemical and chromatographic analyses, MS and conductometric measurements, as well as by IR and NMR (1H and 13C) spectral studies. The crystal structures of ligand and mononuclear complex, trans-dichlorobis(2,6-dimethyl-4-nitro-pyridine)palladium(II), were determined by three-dimensional X-ray methods. The crystals of both compounds are monoclinic, space groups P21/c with a=19.075(4), b=5.419(1), c=15.045(3) A and beta=108.15(3)degrees for (dmnp), and a=7.544(2), b=14.509(3), c=8.032(2) A and beta=90.32(3)degrees for [Pd(dmnp)2Cl2]. In the (dmnp) there are two crystallographically independent molecules in the unit cell. The nitro groups and methyl C atoms are coplanar with the ring plane. The hydrogen bond of the type C-H...O links the molecules into pairs around center of symmetry. These dimers are held together by contacts of the van der Waals type. In the crystal structure of [Pd(dmnp)2Cl2] the Pd atom lies on an inversion center and is four-coordinated by two pyridine N atoms and by two Cl atoms in trans positions. The coordination geometry is square-planar, with Pd-N and Pd-Cl distances of 2.033(2) and 2.311(1) A, respectively. The two pyridine rings are mutually parallel, but they are twisted from the PdN2Cl2 coordination plane by about 88.5degrees. The preliminary assessments of anti-tumor properties of both complexes and ligand were evaluated as in vitro anti-proliferative activity in four human cancer cell lines: SW707 (adenocarcinoma of the rectum), T47D (breast cancer), HCV (bladder cancer) and A549 (non-small cell lung carcinoma). The [Pd(dmnp)2Cl2] exhibits strong cytotoxic activity against all cell lines whereas the free ligand and dinuclear [Pd2(dmnp)2Cl4] are only moderate active.     

Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.     

Two-dimensional crystallization of a small heat shock protein HSP16.3 on lipid layer

As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.     

Cobalt substitution of mouse R2 ribonucleotide reductase as a model for the reactive diferrous state: spectroscopic and structural evidence for a ferromagnetically coupled dinuclear cobalt cluster

The R2 dimer of mouse ribonucleotide reductase contains a dinuclear iron-oxygen cluster and tyrosyl radical/subunit. The dinuclear diferrous form reacts with dioxygen to generate the tyrosyl radical essential for the catalytic reaction that occurs at the R1 dimer. It is important to understand how the reactivity toward oxygen is related to the crystal structure of the dinuclear cluster. For the mouse R2 protein, no structure has been available with a fully occupied dinuclear metal ion site. A cobalt substitution of mouse R2 was performed to produce a good model for the very air-sensitive diferrous form of the enzyme. X-band EPR and light absorption studies (epsilon(550 nm) = 100 mm(-1) cm(-1)/Co(II)) revealed a strong cooperative binding of cobalt to the dinuclear site. In perpendicular mode EPR, the axial signal from mouse R2 incubated with Co(II) showed a typical S = 3/2 Co(II) signal, and its low intensity indicated that the majority of the Co(II) bound to R2 is magnetically coupled. In parallel mode EPR, a typical integer spin signal (M(s) = +/-3) with g approximately 12 is observed at 3.6 K and 10 K, showing that the two Co(II) ions (S = 3/2) in the dinuclear site are ferromagnetically coupled. We have solved the 2.4 A crystal structure of the Co(II)-substituted R2 with a fully occupied dinuclear cluster. The bridging Co(II) carboxylate ligand Glu-267 adopts an altered orientation compared with its counterpart Glu-238 in Escherichia coli R2. This might be important for proper O(2) activation of the more exposed native diferrous site in mouse R2 compared with E. coli R2.     

Engineering of betabellin 15D: Copper(II)-induced folding of a fibrillar β-sandwich protein

The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue -sandwich protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium acetate with and without Cu2+. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited -sheet structure in the presence of Cu2+ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence of Cu2+ but not in its absence. The observed width of the fibrils (7 ± 2 nm) was consistent with the width (6.8 nm) of a structural model of a fibril that contained two adjacent rows of betabellin 15D -sandwiches joined lengthwise by multiple intersheet hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural model of the Cu(II)/betabellin 15D fibril.     

Studies on the active site of deacetoxycephalosporin C synthase

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; alpha=beta=90 degrees, gamma=120 degrees (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.     

Crystal structure of the catalytic domain of human bile salt activated lipase

Bile-salt activated lipase (BAL) is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1-538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 A resolution. The crystal form belongs to space group P2(1)2(1)2(1) with one monomer per asymmetric unit, and the protein shows an alpha/beta hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115-125 and 270-285).     

Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67

Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.