Preliminary x-ray crystallographic analysis of centrin from ciliate Euplotes octocarinatus

Centrins are four-EF-hand Ca(2+)-binding proteins, which belong to the CaM super family. The centrin from ciliate Euplotes Octocarinatus has been expressed in Escherichia coli, purified and crystallized using the hanging-drop method. Rod-like crystals were grown and diffracted to 2.0 angstroms. The crystals belong to space group P2(1)2(1)2(1) and the unit-cell parameters are a=34.442 angstroms, b=48.954 angstroms, c=72.583 angstroms.     

GTPase activity analysis of eRF3 in Euplotes octocarinatus

In eukaryotes, eRF3 participates translation termination and belongs to the superfamily of GTPase. In this work, dissociation constants for E. octocarinatus eRF3 binding to nucleosides in presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, the GTP hydrolyzing assay of Eo-eRF3 was carried out by HPLC methods and the kinetic parameter for GTP hydrolysis by eRF3 was determined. The results showed eRF1a could promote GTP binding to eRF3 and hydrolyzing GTP activity of eRF3. The observation is consistent with the data from human. Whereas E. octocarinatus eRF3 alone can bind GTP in contrast to no GTP binding observed in the absence of eRF1 in human eRF3. The affinity for Eo-eRF3 binding nucleotides is different from that in human. Structure model and amino acids sequence alignment of potential G domains indicated these different may be due to Valine 317 and Glutamate 452 displacing conserved Glycine and Lysine, which were involved in GTP binding.     

eRF1aMC and Mg(2+) dependent structure switch of GTP binding to eRF3 in Euplotes octocarinatus

Eukaryotic translation termination is governed by eRF1 and eRF3. eRF1 recognizes the stop codons and then hydrolyzes peptidyl-tRNA. eRF3, which facilitates the termination process, belongs to the GTPase superfamily. In this study, the effect of the MC domain of eRF1a (eRF1aMC) on the GTPase activity of eRF3 was analyzed using fluorescence spectra and high-performance liquid chromatography. The results indicated eRF1aMC promotes the GTPase activity of eRF3, which is similar to the role of eRF1a. Furthermore, the increased affinity of eRF3 for GTP induced by eRF1aMC was dependent on the concentration of Mg(2+). Changes in the secondary structure of eRF3C after binding GTP/GDP were detected by CD spectroscopy. The results revealed changes of conformation during formation of the eRF3C·GTP complex that were detected in the presence of eRF1a or eRF1aMC. The conformations of the eRF3C·eRF1a·GTP and eRF3C·eRF1aMC·GTP complexes were further altered upon the addition of Mg(2+). By contrast, there was no change in the conformation of GTP bound to free eRF3C or the eRF3C·eRF1aN complex. These results suggest that alterations in the conformation of GTP bound to eRF3 is dependent on eRF1a and Mg(2+), whereas the MC domain of eRF1a is responsible for the change in the conformation of GTP bound to eRF3 in Euplotes octocarinatus.     

Lutetium(III)-dependent self-assembly study of ciliate Euplotes octocarinatus centrin

Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins, which often associated with the centrosomes and basal bodies. To explore the possible structural role of EoCen, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. The native PAGE results indicate that only the integral protein shows multimers in the presence of Lu(3+). The dependence of Lu(3+) induced self-assembly of EoCen on various chemical and physical factors, including temperature, protein concentration, ionic strength and pH, was characterized using resonance light scattering (RLS). Control experiments with different metal ions suggest that Ca(2+) and Lu(3+) bindings to the N-terminal domain of EoCen are all positive to the self-assembly of the protein, and Lu(3+) exhibits the stronger effect, however, Mg(2+) alone cannot take the same effect. The experiments of 2-ptoluidinylnaphthalene-6-sulfonate (TNS) binding and ionic strength demonstrate that the lutetium(III)-dependent self-assembly is closely related to the exposure of hydrophobic cavity. Control experiment on pH value with EoCen and the fragments of it, N-terminal domain of EoCen (N-EoCen), indicates that the electrostatic effect is of small tendency to be served as the main driving force in the self-assembly of EoCen. The specific oligomerization form of the protein was exhibited by cross-linking experiment.     

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.     

Class-1 release factor eRF1 promotes GTP binding by class-2 release factor eRF3

In eukaryotes, termination of mRNA translation is triggered by the essential polypeptide chain release factors eRF1, recognizing all three stop codons, and eRF3, a member of the GTPase superfamily with a role that has remained opaque. We have studied the kinetic and thermodynamic parameters of the interactions between eRF3 and GTP, GDP and the non-hydrolysable GTP analogue GDPNP in the presence (K(D)(GDP)=1.3+/-0.2 muM, K(D)(GTP) approximately 200 muM and K(D)(GDPNP)>160 muM) as well as absence (K(D)(GDP)=1.9+/-0.3 muM, K(D)(GTP) 0.7+/-0.2 muM and K(D)(GDPNP) approximately 200 muM) of eRF1. From the present data we propose that (i) free eRF3 has a strong preference to bind GDP compared to GTP (ii) eRF3 in complex with eRF1 has much stronger affinity to GTP than free eRF3 (iii) eRF3 in complex with PABP has weak affinity to GTP (iv) eRF3 in complex with eRF1 does not have strong affinity to GDPNP, implying that GDPNP is a poor analogue of GTP for eRF3 binding.     

Preliminary X-ray crystallographic analysis of intercellular adhesion molecule-1.

Crystals of the two amino-terminal domains of intercellular adhesion molecule-1, the receptor for the major group of human rhinovirus serotypes, diffract to 3.0 A resolution. The crystals are trigonal in space group P3(1)21 or P3(2)21 with cell dimensions of a = b = 55.7 A, c = 166.3 A, with probably six molecules per unit cell.     

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).     

Effects of food availability on predator-induced morphological defence in the ciliate Euplotes octocarinatus (Protista)

The effect of a prey's food resources on predator-induced morphological transformation was studied in two ciliate species: Euplotes octocarinatus (prey) and Stylonychia mytilus (predator). The ability to produce defence in Euplotes was much reduced by prolonged starvation. The extent of prey morphological transformation and therefore the effectiveness of the defence was inversely related to the length of time without food. The results suggest that the defence involves an energetic cost which may be a significant part of the whole cell budget, at least when food is in short supply. Not only the morphological transformation but also the maintenance of the anti-predator phenotype were energetically costly for Euplotes, the cost being proportional to the defence level. The morphological transformation was affected by the kind of food (algae) provided to the prey. Ciliates fed Chlorogonium elongatum attained their maximum width sooner than those fed Chlamydomonas. Thus, both the quantity and the quality of the food available may significantly affect the magnitude of response and therefore the potential success of an induced defence.     

Gamone Production in Large-Scale Cultures of Euplotes octocarinatus1

We report a method that allows us to grow and maintain the freshwater ciliate Euplotes octocarinatus in large quantities. Frequent exchange of culture fluid proved more effective than aeration in obtaining high cell densities (4200 cells/ml) and reasonable doubling times in large-scale cultures. For harvesting gamone 1, the cell density was raised to 10,000 cells/ml. Under these conditions, the cells continued to produce and secrete gamone; they were slightly starved, but they no longer divided. Cell-free fluid with a steady and relatively high yield of gamone was obtained from two such cultures over a period of five months. We isolated gamone 1 also from cell homogenates and compared it with secreted gamone 1, but found no differences in the gamones from these two sources.     

Pheromone 4 gene of Euplotes octocarinatus

We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5′ and 3′ splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3′ end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macro-nuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes. © 1992 Wiley-Liss, Inc.     

How translation termination factor eRF1 Euplotes does not recognise UGA stop codon

In universal-code eukaryotes, a single class-1 translation termination factor eRF1 decodes all three stop codons, UAA, UAG, and UGA. In some ciliates with variant genetic codes one or two stop codons are used to encode amino acid(s) and are not recognized by eRF1. In Stylonychia, UAG and UAA codons are reassigned as glutamine codons, and in Euplotes, UGA is reassigned as cysteine codon. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of eRF1. Because variant-code ciliates most likely evolved from universal code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. To find out amino acid residues which confer UAR-only specificity to Euplotes aediculatus eRF1, eRFI chimeras were constructed by swapping eRF1 E. aediculatus N-terminal domain sequences with the matching ones from the human protein. In these chimeras the MC-domain was from human eRF1. Functional analysis of these chimeric eRFI highlighted the crucial role of the two regions (positions 38-50 and 123-145) in the N-terminal domain of E. aediculatus eRF1 that restrict E. aediculatus eRF1 specificity toward UAR codons. Possibly, restriction of eRF1 specificity to UAR codons might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UGA to sense codons.     

Isolation and partial characterization of gamone 1 of Euplotes octocarinatus

A polypeptide, termed gamone 1, was isolated and purified to homogeneity from culture filtrates of mating type VII of the freshwater ciliate Euplotes octocarinatus. The gamone induces intraclonal conjugation in cells of certain other mating types. The isolation and purification of the gamone was carried out by a combination of two chromatographic steps. The purified gamone was found to be still effective in a concentration of approx. 10⁻¹⁵M.     

Preliminary X-ray crystallographic analysis of holotoxin from Bordetella pertussis

Pertussis (whooping cough) is a serious infectious disease caused by the bacterium Bordetella pertussis. One of the major virulence factors is a protein known as pertussis toxin, which is composed of six subunits, with a total molecular weight of 106,000. Enzymatic transfer of ADP-ribose from NAD to a family of GTP-binding proteins is effected by the largest subunit (S1 or the A monomer), while binding of host cells and entry of S1 to the interior is a function of the other subunits (the B oligomer). The holotoxin crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 98.4 A, b = 164.2 A and c = 195.2 A. The crystals are suitable for high-resolution X-ray diffraction analysis.     

Pheromone 4 gene of Euplotes octocarinatus.

We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5' and 3' splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3' end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macronuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes.     

八肋游仆虫核糖体蛋白L11的基因克隆与序列分析

以单细胞真核生物八肋游仆虫Euplotes octocarinatus为实验材料,采用PCR、RT-PCR方法克隆了核糖体蛋白L11基因(EoRPL11).将该序列与GenBank中其他物种的RPL11基因序列进行同源性比对,采用DNAStar软件进行聚类分析.结果显示,成功克隆到游仆虫RPL11基因,大核中该基因全长709 bp,开放阅读框(ORF)为531 bp,编码176个氨基酸;cDNA序列与大核中ORF序列一致,表明该基因具有转录活性;所推导的氨基酸序列与嗜热四膜虫Tetrahymena thermophila的RPL11同源性最高,为66%.     

Preliminary crystallographic analysis of a plant pathogenic factor: pectate lyase

Pectate lyase is a saccharide-binding enzyme that lyitically depolymerizes polypectate in higher plant cell walls, thus causing soft-rot diseases in food crops. A pectate lyase from Erwinia chrysanthemi, EC16 (PLe), crystallizes in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimension of a = 39.0 A, b = 91.0 A and c = 103.4 A. The asymmetric unit consists of one molecule with a molecular mass of 38,118 daltons and the X-ray diffraction extends to a resolution of 1.8 A. The crystals reproducibly grow to large dimensions and are suitable for a high-resolution X-ray diffraction analysis.     

Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase.

Termination of translation in eukaryotes is governed by two polypeptide chain release factors, eRF1 and eRF3 on the ribosome. eRF1 promotes stop-codon-dependent hydrolysis of peptidyl-tRNA, and eRF3 interacts with eRF1 and stimulates eRF1 activity in the presence of GTP. Here, we have demonstrated that eRF3 is a GTP-binding protein endowed with a negligible, if any, intrinsic GTPase activity that is profoundly stimulated by the joint action of eRF1 and the ribosome. Separately, neither eRF1 nor the ribosome display this effect. Thus, eRF3 functions as a GTPase in the quaternary complex with ribosome, eRF1, and GTP. From the in vitro uncoupling of the peptidyl-tRNA and GTP hydrolyses achieved in this work, we conclude that in ribosomes both hydrolytic reactions are mediated by the formation of the ternary eRF1-eRF3-GTP complex. eRF1 and the ribosome form a composite GTPase-activating protein (GAP) as described for other G proteins. A dual role for the revealed GTPase complex is proposed: in " GTP state," it controls the positioning of eRF1 toward stop codon and peptidyl-tRNA, whereas in "GDP state," it promotes release of eRFs from the ribosome. The initiation, elongation, and termination steps of protein synthesis seem to be similar with respect to GTPase cycles.