The structure of the near germline antibody S25-2 in complex with an unnatural Kdo disaccharide highlights the remarkable plasticity in antigen recognition of germline antibodies.
The near-germline antibody S25-2 exhibits a remarkable cross-reactivity for oligosaccharides containing the bacterial lipopolysaccharide carbohydrate 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). The recent synthesis of a variety of Kdo analogues permits a detailed structural analysis of the importance of specific interactions in antigen recognition by S25-2. The Kdo disaccharide analogue Kdo-(2→4)-5,6-dehydro-Kdo lacks a 5-OH group on the second Kdo residue and has been cocrystallized with S25-2. The structure reveals that the modification of the Kdo residue at position 5 results in a rearrangement of intramolecular hydrogen bonds in the antigen that allows it to assume a novel conformation in the antibody-combining site. The cross-reactive binding of S25-2 to this synthetic ligand highlights the adaptability of this antibody to non-natural synthetic analogues.
antibody–antigen interactions; protein–carbohydrate interactions; cross-reactive binding
The crystallization and structure determination to 2.2 Å resolution is reported for a ribulose 1,5-bisphosphate-bound non-activated form of garden pea ribulose-1,5-bisphosphate carboxylase/oxygenase.
The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 Å resolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the ‘closed’ conformation observed in other substrate/inhibitor-bound Rubisco structures.
Pisum sativum; Rubisco; molecular replacement; ribulose 1,5-bisphosphate
The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the ectodomain of BVDV E2 are described.
Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.
Pestivirus; BVDV; envelope glycoprotein E2
The crystallization and preliminary X-ray crystallographic analysis of diaminopimelate epimerase from A. baumannii are reported.
The meso isomer of diaminopimelate (meso-DAP) is a biosynthetic precursor of l-lysine in bacteria and plants, and is a key component of the peptidoglycan layer in the cell walls of Gram-negative and some Gram-positive bacteria. Diaminopimelate epimerase (DapF) is a pyridoxal-5′-phosphate-independent racemase which catalyses the interconversion of (6S,2S)-2,6-diaminopimelic acid (ll-DAP) and meso-DAP. In this study, DapF from Acinetobacter baumannii was overexpressed in Escherichia coli strain SoluBL21, purified and crystallized using a vapour-diffusion method. A native crystal diffracted to a resolution of 1.9 Å and belonged to space group P31 or P32, with unit-cell parameters a = b = 74.91, c = 113.35 Å, α = β = 90, γ = 120°. There were two molecules in the asymmetric unit.
meso-diaminopimelate; diaminopimelate epimerase; Acinetobacter baumannii
A mutant human MTH1 protein [hMTH1(G2K)] with a homogeneous N-terminus produced high-quality crystals which diffracted to near 1.1 Å resolution using synchrotron radiation.
Human MTH1 (hMTH1) is an enzyme that hydrolyses several oxidized purine nucleoside triphosphates to their corresponding nucleoside monophosphates. Crystallographic studies have shown that the accurate mode of interaction between 8-oxoguanine and hMTH1 cannot be understood without determining the positions of the H atoms, as can be observed in neutron and/or ultrahigh-resolution X-ray diffraction studies. The hMTH1 protein prepared in the original expression system from Escherichia coli did not appear to be suitable for obtaining high-quality crystals because the hMTH1 protein had heterogeneous N-termini of Met1 and Gly2 that resulted from N-terminal Met excision by methionine aminopeptidase from the E. coli host. To obtain homogeneous hMTH1, the Gly at the second position was replaced by Lys. As a result, mutant hMTH1 protein [hMTH1(G2K)] with a homogeneous N-terminus could be prepared and high-quality crystals which diffracted to near 1.1 Å resolution using synchrotron radiation were produced. The new crystals belonged to space group P212121, with unit-cell parameters a = 46.36, b = 47.58, c = 123.89 Å.
The hypothetical protein MJ0927 of the Nif3 family has been crystallized and X-ray diffraction data have been collected to a resolution of 2.47 Å.
MJ0927 is a member of the Nif3 family and is widely distributed across living organisms. Although several crystal structures of Nif3 proteins have been reported, structural information on archaeal Nif3 is still limited. To understand the structural differences between bacterial and archaeal Nif3 proteins, MJ0927 from Methanocaldococcus jannaschii was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.47 Å and belonged to the orthorhombic space group C222, with unit-cell parameters a = 81.21, b = 172.94, c = 147.42 Å. Determination of this structure may provide insights into the function of MJ0927.
Nif3; Methanocaldococcus jannaschii; metal-binding proteins
The preliminary crystallographic analysis of Megavirus chilensis Mg561, which has a predicted polyadenylate synthase function, is reported. The crystals belonged to space group P212121, with two monomers per asymmetric unit.
Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Å resolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF.
Megavirus chilensis; Megaviridae; giant viruses; transcription; poly(A) polymerase; polyadenylate synthase
Crystals of the phosphotriesterase from M. tuberculosis were obtained and diffraction data were collected and processed to 2.27 Å resolution. An analytical ultracentrifugation experiment suggested that mPHP exists as dimers in solution.
Organophosphates (OPs) are extremely toxic compounds that are used as insecticides or even as chemical warfare agents. Phosphotriesterases (PHPs) are responsible for the detoxification of OPs by catalysing their degradation. Almost 100 PHP structures have been solved to date, yet the crystal structure of the phosphotriesterase from Mycobacterium tuberculosis (mPHP) remains unavailable. This study reports the first crystallization of mPHP. The crystal belonged to space group C2221, with unit-cell parameters a = 68.03, b = 149.60, c = 74.23 Å, α = β = γ = 90°. An analytical ultracentrifugation experiment suggested that mPHP exists as a dimer in solution, even though one molecule is calculated to be present in the asymmetric unit according to the structural data.
Mycobacterium tuberculosis; phosphotriesterases
D. melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization and preliminary X-ray crystallographic analysis of the complex of the two proteins are reported.
Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source.
Drosophila melanogaster; Gαo-subunit; G-protein signalling
The UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (VvGT5) from the grapevine V. vinifera was purified and crystallized. The best crystal diffracted X-rays to 2.2 Å resolution and belonged to space group P6122.
Grapevine (Vitis vinifera) glycosyltransferase 5 (VvGT5) is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase that catalyses the 3-O-specific glucuronosylation of flavonols using UDP-glucuronic acid as a sugar donor to produce flavonol 3-O-glucosides, which are important bioactive phytochemicals. Recombinant VvGT5 expressed in Escherichia coli cells was purified and crystallized by the sitting-drop vapour-diffusion method. A full set of X-ray diffraction data was collected to 2.2 Å Bragg spacing from a single crystal using a synchrotron-radiation source. The crystal was hexagonal, belonging to space group P6122, with unit-cell parameters a = b = 102.70, c = 535.92 Å. The initial phases were determined by the molecular-replacement method.
VvGT5; UGT; glycosyltransferase; flavonol; flavonoid
Crystals of Deg8, an ATP-independent serine endopeptidase from A. thaliana, were monoclinic, belonging to space group C2 with unit-cell parameters a = 129.5, b = 124.2, c = 93.3 Å, β = 132.4°, and diffracted to 2.0 Å resolution.
Arabidopsis thaliana Deg8, an ATP-independent serine endopeptidase, is involved in the repair of photosystem II (PSII), specifically the degradation of the photo-damaged PSII reaction centre D1 protein. To understand the molecular mechanism underlying the participation of Deg8 in the degradation of the photo-damaged D1 protein, the structure of Deg8 is needed. Until recently, however, no structure of Deg8 had been solved. In this study, Deg8 from A. thaliana was cloned, overexpressed and purified in Escherichia coli. Crystallization was performed at 277 K using tribasic sodium citrate as the precipitant and the crystals diffracted to 2.0 Å resolution, belonging to space group C2 with unit-cell parameters a = 129.5, b = 124.2, c = 93.3 Å, α = γ = 90, β = 132.4°. Assuming one trimer in the asymmetric unit, the Matthews coefficient and the solvent content were calculated to be 2.35 Å3 Da−1 and 47.6%, respectively.
Deg8; photosystem II; Arabidopsis thaliana
An organophosphorus hydrolase from P. pseudoalcaligenes named OPHC2 has been crystallized. Combined with biochemical characterization, it is expected that the structure of this protein will provide insight into the catalytic mechanism of organophosphorus hydrolysis and will highlight the role of key residues involved in substrate specificity.
Enzymes that are capable of degrading neurotoxic organophosphorus compounds are of increasing interest because of the lack of efficient and clean methods for their removal. Recently, a novel organophosphorus hydrolase belonging to the metallo-β-lactamase superfamily was identified and isolated from the mesophilic bacterium Pseudomonas pseudoalcaligenes. This enzyme, named OPHC2, is endowed with significant thermal and pH stability, making it an appealing candidate for engineering studies to develop an efficient organophosphorus biodecontaminant. Combined with biochemical studies, structural information will help decipher the catalytic mechanism of organophosphorus hydrolysis by OPHC2 and identify the residues involved in its substrate specificity. Here, the expression, purification, crystallization and X-ray data collection at 2.1 Å resolution of OPHC2 are presented.
organophosphorus; quorum sensing; phosphotriesterases; lactonases; nerve agents; pesticides; quorum quenching; Pseudomonas pseudoalcaligenes
A bacteria biofilm formation involved enzyme, BsYisP, from Bacillus subtilis subsp. subtilis strain 168, was crystallized and diffracted to 1.92 Å.
YisP is an enzyme involved in the pathway of biofilm formation in bacteria and is predicted to possess squalene synthase activity. A BlastP search using the YisP protein sequence from Bacillus subtilis subsp. subtilis strain 168 shows that it shares 23% identity with the dehydrosqualene synthase from Staphylococcus aureus. The YisP from B. subtilis 168 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. The crystals, which belong to the orthorhombic space group P212121, with unit-cell parameters a = 43.966, b = 77.576, c = 91.378 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.92 Å resolution. Structure determination using MAD and MIR methods is in progress.
YisP; squalene synthase; dehydrosqualene synthase; Bacillus subtilis subsp. subtilis; drug design; synchrotron radiation
Europium(III) ions bound to the surface of hen egg-white lysozyme were found to exhibit good anomalous signal facilitating SAD phasing using laboratory-source data and automated model building. The europium ion-binding sites were observed up to the 15σ level.
Europium is shown to be a good anomalous scatterer in SAD phasing for solving the structure of biological macromolecules. The large value of the anomalous contribution of europium, f′′ = 11.17 e−, at the Cu Kα wavelength is an advantage in de novo phasing and automated model building. Tetragonal crystals of hen egg-white lysozyme (HEWL) incorporating europium(III) chloride (50 mM) were obtained which diffracted to a resolution of 2.3 Å at a wavelength of 1.54 Å (Cu Kα). The master data set (360° frames) was split and analyzed for anomalous signal-to-noise ratio, multiplicity, completeness, SAD phasing and automated building. The structure solution and model building of the split data sets were carried out using phenix.autosol and phenix.autobuild. The contributions of the Eu ions to SAD phasing using in-house data collection are discussed. This study revealed successful lysozyme phasing by SAD using laboratory-source data involving Eu ions, which are mainly coordinated by the side chains of Asn46, Asp52 and Asp101 together with some water molecules.
europium; SAD; lysozyme
Crystal structures of the hexanucleotide d(CACGCG)·d(CGCGTG) were determined in the presence of Mn2+ ions in two crystal lattices and provide insights into ion interactions.
Crystal structures of the hexanucleotide d(CACGCG)·d(CGCGTG) were determined in two crystal lattices when different concentrations of the counterion Mn2+ were used in crystallization. The availability of Mn2+ during the crystallization process appears to play an important role in inducing different crystal packings that lead to crystals belonging to the two space groups P21 and P65. Analysis of the molecular interactions of Mn2+ with the Z-form duplexes shows direct coordination to the purine residues G and A.
Z-DNA; crystal packing; DNA–ion interactions; manganese ions
The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 was determined at a resolution of 3.02 Å, showing a new relative arrangement of the OB-fold domain.
DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5′-phosphate and 3′-hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Å shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.
ATP-dependent DNA ligase; OB fold; Thermococcus sp. 1519; DNA ligases
A 118 kDa fragment, comprising the catalytic domain and four other domains, of the glucansucrase GTFA from L. reuteri 121, which synthesizes α-glucans with both α-1,6- and α-1,4-glycosidic linkages, was crystallized. The weakly diffracting crystals, which contained 85% solvent, were used to determine the structure at 3.6 Å resolution.
The reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched α-glucans with both α-1,6- and α-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-ΔN, a 118 kDa fragment of GTFA comprising residues 745–1763 and including the catalytic domain, was determined at 3.6 Å resolution by molecular replacement. The crystals have large solvent channels and an unusually high solvent content of 85%. GTFA-ΔN has the same domain arrangement and domain topologies as observed in previously determined GH70 glucansucrase structures. The architecture of the GTFA-ΔN active site and binding pocket confirms that glucansucrases have a conserved substrate specificity for sucrose. However, this first crystal structure of an α-1,6/α-1,4-specific glucansucrase shows that residues from conserved sequence motif IV (1128–1136 in GTFA-ΔN) contribute to the acceptor-binding subsites and that they display differences compared with other structurally characterized glucansucrases. In particular, the structure clarifies the importance of residues following the transition-state stabilizer for product specificity, and especially residue Asn1134, which is in a position to interact with sugar units in acceptor subsite +2.
lactic acid bacteria; glucansucrase; reuteransucrase
The Xpln crystal structure provides structural insights into Rho GTPase binding.
Xpln is a guanine nucleotide-exchange factor (GEF) for Rho GTPases. A Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain is a widely adopted GEF-domain architecture. The Xpln structure solely comprises these two domains. Xpln activates RhoA and RhoB, but not RhoC, although their GTPase sequences are highly conserved. The molecular mechanism of the selectivity of Xpln for Rho GTPases is still unclear. In this study, the crystal structure of the tandemly arranged DH-PH domains of mouse Xpln, with a single molecule in the asymmetric unit, was determined at 1.79 Å resolution by the multiwavelength anomalous dispersion method. The DH-PH domains of Xpln share high structural similarity with those from neuroepithelial cell-transforming gene 1 protein, PDZ-RhoGEF, leukaemia-associated RhoGEF and intersectins 1 and 2. The crystal structure indicated that the α4–α5 loop in the DH domain is flexible and that the DH and PH domains interact with each other intramolecularly, thus suggesting that PH-domain rearrangement occurs upon RhoA binding.
GEF proteins; DH-PH module structure
Inorganic pyrophosphatase from T. thioreducans has been crystallized and the crystals were deemed to be suitable for both X-ray and neutron diffraction at room temperature.
Inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens was cloned, overexpressed in Escherichia coli, purified and crystallized in restricted geometry, resulting in large crystal volumes exceeding 5 mm3. IPPase is thermally stable and is able to resist denaturation at temperatures above 348 K. Owing to the high temperature tolerance of the enzyme, the protein was amenable to room-temperature manipulation at the level of protein preparation, crystallization and X-ray and neutron diffraction analyses. A complete synchrotron X-ray diffraction data set to 1.85 Å resolution was collected at room temperature from a single crystal of IPPase (monoclinic space group C2, unit-cell parameters a = 106.11, b = 95.46, c = 113.68 Å, α = γ = 90.0, β = 98.12°). As large-volume crystals of IPPase can be obtained, preliminary neutron diffraction tests were undertaken. Consequently, Laue diffraction images were obtained, with reflections observed to 2.1 Å resolution with I/σ(I) greater than 2.5. The preliminary crystallographic results reported here set in place future structure–function and mechanism studies of IPPase.
inorganic pyrophosphatase; Thermococcus thioreducens; neutron diffraction
Crystals of S. scrofa quinolinate phosphoribosyltransferase purified from porcine kidney in complex with nicotinate mononucleotidewere obtained and diffraction data were collected and processed to 2.1 Å resolution.
Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Å resolution. The Ss-QAPRTase–NAMN cocrystals belonged to space group P321, with unit-cell parameters a = 119.1, b = 119.1, c = 93.7 Å, γ = 120.0°. The Matthews coefficient and the solvent content were estimated as 3.10 Å3 Da–1 and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit.
quinolinate phosphoribosyltransferase; NAD biosynthesis; nicotinate mononucleotide
The enzyme porphobilinogen deaminase (PBGD) catalyses a key early step in the biosynthesis of haem in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. PBGD from the model plant organism A. thaliana has been expressed and the enzyme was crystallized in a form that diffracted synchrotron radiation to high resolution.
The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 126.96.36.199) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution.
tetrapyrrole biosynthesis; porphobilinogen deaminase; dipyrromethane complex
The cloning, expression, purification and crystallization of phosphoethanolamine transferase A, an endotoxin-modifying enzyme from N. meningitidis, are reported.
The enzyme phosphoethanolamine transferase A is involved in the addition of phosphoethanolamine moieties to lipid A in Neisseria meningitidis. The enzyme is composed of an N-terminal transmembrane domain and a C-terminal soluble domain that is present in the periplasm of the bacteria. A membrane-deletion construct of the enzyme was designed and expressed in Escherichia coli. Well ordered crystals that diffracted to 1.7 Å resolution were obtained by carrying out a limited trypsin digestion of the protein to remove a predicted N-terminal disordered portion. The crystals belonged to space group P21, with unit-cell parameters a = 44.3, b = 71.6, c = 49.9 Å, β = 109.2°, and contained one molecule in the asymmetric unit.
endotoxin biosynthesis; LptA; phosphoethanolamine transferase
The PH domain and ORD of the oxysterol-binding protein Osh3 from S. cerevisae were crystallized and X-ray diffraction data were collected.
Oxysterol-binding protein (OSBP) related proteins (ORPs) are conserved from yeast to humans and are implicated in regulation of sterol homeostasis and in signal transduction pathways. Osh3 of Saccharomyces cerevisiae is a pleckstrin-homology (PH) domain-containing ORP member that regulates phosphoinositide metabolism at endoplasmic reticulum–plasma membrane contact sites. The N-terminal PH domain of Osh3 was purified and crystallized as a lysozyme fusion and the resulting crystal diffracted to 2.3 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 98.03, b = 91.31, c = 84.13 Å, β = 81.41°. With two molecules in the asymmetric unit, the Matthews coefficient was 3.13 Å3 Da−1. Initial attempts to solve the structure by molecular-replacement techniques using T4 lysozyme as a search model were successful. The C-terminal OSBP-related domain (OBD) of Osh3 was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 1.5 Å resolution. The crystal was orthorhombic, belonging to space group P212121, with unit-cell parameters a = 41.57, b = 87.52, c = 100.58 Å. With one molecule in the asymmetric unit, the Matthews coefficient was 2.01 Å3 Da−1. Initial attempts to solve the structure by the single-wavelength anomalous dispersion technique using bromine were successful.
oxysterol-binding protein; Osh3; Saccharomyces cerevisiae
A novel stage-specific surface protein, T. congolense insect-stage surface antigen (TcCISSA), from the African trypanosome T. congolense was recently identified by mass-spectrometric differential protein-expression analysis. To gain structure–function insight into this protein, the extracellular domain of TcCISSA was expressed, purified and crystallized and X-ray diffraction data were collected and processed to 2.7 Å resolution.
Trypanosoma congolense is a major contributor to the vast socioeconomic devastation in sub-Saharan Africa caused by animal African trypanosomiasis. These protozoan parasites are transmitted between mammalian hosts by tsetse-fly vectors. A lack of understanding of the molecular basis of tsetse–trypanosome interactions stands as a barrier to the development of improved control strategies. Recently, a stage-specific T. congolense protein, T. congolense insect-stage surface antigen (TcCISSA), was identified that shows considerable sequence identity (>60%) to a previously identified T. brucei insect-stage surface molecule that plays a role in the maturation of infections. TcCISSA has multiple di-amino-acid and tri-amino-acid repeats in its extracellular domain, making it an especially interesting structure–function target. The predicted mature extracellular domain of TcCISSA was produced by recombinant DNA techniques, purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis; the data were processed to 2.7 Å resolution.
Trypanosoma congolense; procyclic form; animal African trypanosomiasis; insect stage; vector–pathogen interactions; surface proteins
Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from F. nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. The crystals obtained were characterized by X-ray diffraction.
Hydrogen sulfide produced by oral bacteria is responsible for oral malodour. Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from Fusobacterium nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. X-ray diffraction data were collected from the crystals using a synchrotron-radiation source. The Fn1220 crystal belonged to tetragonal space group P41212 or P43212 (unit-cell parameters a = b = 116.8, c = 99.2 Å) and the Cdl crystal belonged to monoclinic space group P21 (unit-cell parameters a = 84.9, b = 70.9, c = 87.6 Å, β = 90.3°).
hydrogen sulfide; Fn1220; Cdl; Fusobacterium nucleatum