Septin crystallization for structural analysis.

Septins are filament-forming proteins found in many eukaryotes. Despite being important components of the cytoskeleton, only recently details of their macromolecular assemblies and crystal structures have started to appear in the literature. These are of fundamental importance to the understanding of cytoskeleton dynamics, membrane barrier formation, and bacterial caging, as well as essential cellular processes such as cell division, exocytosis, and vesicle trafficking. However, obtaining this data is frequently hindered by several experimental difficulties common to the majority of septin samples. Here we provide an overview of the current approaches to circumvent or minimize the experimental complications observed in septin crystallography focusing mainly, but not exclusively, on the choice of the septin construct and how to best prepare the sample itself.

Crystallization and preliminary structural analysis of the giant haemoglobin from Glossoscolex paulistus at 3.2 Å.

Glossoscolex paulistus is a free-living earthworm encountered in south-east Brazil. Its oxygen transport requirements are undertaken by a giant extracellular haemoglobin, or erythrocruorin (HbGp), which has an approximate molecular mass of 3.6 MDa and, by analogy with its homologue from Lumbricus terrestris (HbLt), is believed to be composed of a total of 180 polypeptide chains. In the present work the full 3.6 MDa particle in its cyanomet state was purified and crystallized using sodium citrate or PEG8000 as precipitant. The crystals contain one-quarter of the full particle in the asymmetric unit of the I222 cell and have parameters of a = 270.8 Å, b = 320.3 Å and c = 332.4 Å. Diffraction data were collected to 3.15 Å using synchrotron radiation on beamline X29A at the Brookhaven National Laboratory and represent the highest resolution data described to date for similar erythrocruorins. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt. This revealed that HbGp belongs to the type I class of erythrocruorins and provided an interpretable initial electron density map in which many features including the haem groups and disulfide bonds could be identified.

Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1.

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 A resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 A. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

Crystal structure of calf spleen purine nucleoside phosphorylase complexed to a novel purine analogue.

The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.

Getting the most out of X-ray home sources.

The structures of a 14 kDa phospholipase, an 18 kDa proteinase inhibitor and a novel glycoside hydrolase with molecular weight 60 kDa were solved using the SAD technique and the effects of the amount of anomalous signal, completeness and redundancy of data on heavy-atom substructure determination, phasing and model building were analyzed. All diffraction data sets were collected on a Cu-anode X-ray home source. The structure of the phospholipase was obtained using the anomalous scattering contribution from its 16 S atoms. Three-dimensional models for the other two macromolecules were obtained using the anomalous contribution of I atoms rapidly incorporated into the crystal through the quick cryo-soaking method of derivatization. These results were used to discuss the application of sulfur- and iodine-SAD approaches in combination with X-ray home sources for high-throughput protein crystal structure solution. The estimates of the anomalous signal from S atoms in the gene products of four genomes are given and the prospects for increasing the anomalous contribution using longer wavelengths (e.g. from a chromium home source) and quick cryo-soaking derivatization are discussed. The possibility of rapidly preparing tangible home-source isomorphous derivatives suggests that this approach might become a valuable tool in the future of post-genomic projects.

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose.

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.

Crystal structure of human phosphoglucose isomerase and analysis of the initial catalytic steps.

The second enzyme in the glycolytic pathway, phosphoglucose isomerase (PGI), catalyses an intracellular aldose-ketose isomerization. Here we describe the human recombinant PGI structure (hPGI) solved in the absence of active site ligands. Crystals isomorphous to those previously reported were used to collect a 94% complete data set to a limiting resolution of 2.1 A. From the comparison between the free active site hPGI structure and the available human and rabbit PGI (rPGI) structures, a mechanism for protein initial catalytic steps is proposed. Binding of the phosphate moiety of the substrate to two distinct elements of the active site is responsible for driving a series of structural changes resulting in the polarisation of the active site histidine, priming it for the initial ring-opening step of catalysis.

Expression and preliminary X-ray diffraction studies of cytosolic Cu,Zn superoxide dismutase from Schistosoma mansoni.

Active cytosolic (CT) Cu,Zn superoxide dismutase from Schistosoma mansoni (SmCTSOD) was recovered after thrombin cleavage of a glutathione-S-transferase linked fusion protein (GST-SmCTSOD) expressed in the presence of the active-site metals. Crystals have been obtained in two space groups, P2(1)2(1)2(1) and P2(1). The former have unit-cell parameters a = 74.64, b = 78.24, c = 95.18 A and typically diffract to 2.2 A. The monoclinic crystals have unit-cell parameters a = 39.27, b = 95.08, c = 78.41 A, beta = 103.55 degrees and diffract to at least 1.55 A. The calculated solvent content of the crystals is compatible with two dimers of SmCTSOD in the asymmetric unit in both cases. Molecular-replacement solutions have been obtained for both crystal forms and show that slight distortions in the crystal packing relate one form to the other.

Crystallization and preliminary X-ray analysis of jararhagin, a metalloproteinase/disintegrin from Bothrops jararaca snake venom.

Jararhagin is a toxic protein, isolated from the venom of the snake Bothrops jararaca, which is composed of a metalloprotease domain coupled to a disintegrin/cysteine-rich domain. It induces local haemorrhage owing to the proteolytic digestion of the basement membrane of capillaries. Jararhagin also cleaves the alpha(2)beta(1) integrin on the surface of platelets, thereby acting as a potent inhibitor of collagen-induced platelet aggregation. Crystals of jararhagin were obtained by the vapour-diffusion technique at 273 K in 200 mM sodium acetate, 100 mM cacodylate buffer pH 6.5 and 30% PEG 8000. Diffraction data have been obtained to a resolution of 2.8 A from a single frozen crystal, which belonged to space group P2(1)2(1)2(1) with unit-cell parameters a = 73.7, b = 100.3, c = 133.4 A. The asymmetric unit contains two jararhagin molecules and has a solvent content of 45%. A molecular-replacement solution has been obtained using a homology-built model based on the crystal structure of acutolysin, a haemorrhagic zinc metalloproteinase from the venom of the snake Agkistrodon acutus; attempts are under way to locate the remaining domains.

Development and evaluation of detection systems for staphylococcal exfoliative toxin A responsible for scalded-skin syndrome.

Staphylococcal scalded-skin syndrome is usually diagnosed clinically by its characteristic exfoliating rash. Isolation of Staphylococcus aureus from the patient further supports the diagnosis. Several detection systems have been developed to determine whether the isolated strain produces exfoliative toxin, but none are routinely available in hospital laboratories. In a novel approach, we used computer models to predict the structure of the exfoliative toxins based on other serine proteases and to identify surface epitopes for the production of antibodies that specifically bound the exfoliative toxin A (ETA) serotype. Several rapid immunologically based diagnostic tests for ETA were developed with these antibodies and compared with existing systems. Our results showed that Western blot analysis using these antibodies was in complete correlation with PCR, which has been validated against the "gold standard" mouse model. On the other hand, the double-antibody enzyme-linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion assay gave unacceptably high false-positive results due to interference by staphylococcal protein A. This problem was successfully overcome by the development of a F(ab')(2) fragment ELISA, which was rapid and reproducible and was as sensitive and specific as PCR and Western blot analysis. The F(ab')(2) fragment ELISA is superior to existing diagnostic systems because it is quantitative, which may be related to the severity of the condition, and can detect amounts of exfoliative toxin in the picogram range directly from serum. This is the first detection system with the potential to confirm the diagnosis of staphylococcal scalded-skin syndrome from a routine blood test within 3 h of presentation.

Crystallization and preliminary crystallographic studies of a phospholipase A2 from the venom of the Brazilian snake Bothrops moojeni.

A phospholipase A(2) purified from the venom of the snake Bothrops moojeni has been crystallized by vapour-diffusion techniques in hanging drops at 291 K. The crystals, which were grown in the absence of Ca(2+), belong to the cubic system, space group P432, with unit-cell parameters a = b = c = 91.86 A, and contain one molecule in the asymmetric unit (V(M) = 2.71 A(3) Da(-1)). X-ray diffraction experiments provide data to 2.35 A resolution collected on a rotating-anode home source at cryogenic temperatures. The structure has been solved via molecular-replacement techniques using a single monomer of the crystallographic structure of the phospholipase from the Western diamondback rattlesnake (Crotalus atrox) as a search model.

Preliminary crystallographic studies of EcTI, a serine proteinase inhibitor from Enterolobium contortisiliquum seeds.

Enterolobium contortisiliquum trypsin inhibitor (EcTI) belongs to the Kunitz family of plant inhibitors, which are widely distributed in nature, especially in plant seeds. EcTI is composed of two polypeptide chains with a total of 174 residues, homologous to other inhibitors from the same family. EcTI crystals, which were obtained with the acupuncture-gel technique, diffract to 2.0 A resolution and belong to space group P2(1), with unit-cell parameters a = 37.12, b = 38.42, c = 54.08 A, beta = 98.08 degrees. Molecular-replacement techniques using Erythrina caffra trypsin inhibitor (PDB code 1tie) as the search model indicate one monomer in the asymmetric unit. The secondary-structure content of EcTI was determined by circular dichroism spectroscopy, yielding values compatible with the expected topology.

Structural basis for low catalytic activity in Lys49 phospholipases A2--a hypothesis: the crystal structure of piratoxin II complexed to fatty acid.

Asp49 plays a fundamental role in supporting catalysis by phospholipases A2 by coordinating the calcium ion which aids in the stabilization of the tetrahedral intermediate. In several myotoxins from the venoms of Viperidae snakes, this aspartic acid is substituted by lysine. The loss of calcium binding capacity by these mutants has become regarded as the standard explanation for their greatly reduced or nonexistent phospholipolytic activity. Here we describe the crystal structure of one such Lys49 PLA2, piratoxin-II, in which a fatty acid molecule is observed within the substrate channel. This suggests that such toxins may be active enzymes in which catalysis is interrupted at the stage of substrate release. Comparison of the present structure with other PLA2s, both active and inactive, identifies Lys122 as one of the likely causes of the increased affinity for fatty acid in Lys49 enzymes. Its interaction with the mainchain carbonyl of Cys29 is expected to lead to hyperpolarization of the peptide bond between residues 29 and 30 leading to an increased affinity for the fatty acid headgroup. This strongly bound fatty acid may serve as an anchor to secure the toxin within the membrane thus facilitating its pathological effects.

Identification of free fatty acids in maize protein bodies and purified alpha zeins by (13)C and (1)H nuclear magnetic resonance.

Zeins, the maize storage proteins, are the most abundant proteins in the corn endosperm, and are synthesized on the rough endoplasmatic reticulum and deposited in discrete organelles called protein bodies. Several authors, using circular dichroism and optical rotatory dispersion, have concluded that these proteins have a high alpha-helical content in alcoholic solution. In this work we have studied these proteins, within the protein bodies themselves and after extraction from the corn grains with 70% ethanol, using NMR (nuclear magnetic resonance) spectroscopy. We conclusively demonstrate the presence of free fatty acids within both the protein bodies and also in the alcohol extracted alpha zeins. We present evidence for a direct interaction between the free fatty acids and the alpha zein proteins within the protein body and suggest possible mechanisms by which such an association has arisen during the evolution of the maize endosperm.

Influence of the histidine tail on the structure and activity of recombinant chlorocatechol 1,2-dioxygenase.

We present two efficient expression systems for the chlorocatechol 1, 2-dioxygenase (CCD) from Pseudomonas putida. In the first, CCD (encoded by the clcA gene) was expressed in the pETCLCA vector with the addition of an N-terminal histidine tail. After purification, the enzyme (CCD 6xHis) was proteolytically cleaved with thrombin to remove the His tail. The CD spectra of the cleaved and uncleaved enzymes present only minor differences, indicative of correct protein folding. However, the activity of CCD 6xHis, over a wide range of pH, was typically five times lower. This may be the result of steric hindrance caused by the histidine tail. These data are consistent with results obtained using an alternative construct employing a vector which produces a protein product devoid of the His tail. These results suggest that the His tail may induce subtle effects close to the active site which compromise the recovery of full biological activity.

Determination of the three-dimensional structure of toxins by protein crystallography.

Protein crystallography has significantly contributed to the development of many areas of biochemical research, particularly in the understanding of phenomena related to molecular recognition. Examples include the formation of enzyme-substrate complexes (and their subsequent catalysis), host cell invasion by viruses, antigen neutralization and peptide display by proteins of the immune system and many others. More recently, protein crystallography has also proved to be of great value in unraveling the molecular basis of many diseases as well as in the development of new drugs for their treatment. The X-ray diffraction technique in the elucidation of macromolecular structures is situated at the interface between the traditional research fields of biology, biochemistry, chemistry and physics where researchers are united by a common interest in the detailed understanding of macromolecule function and its relationship to three-dimensional structure. The purpose of this review is to describe, without resort to mathematical detail, all of the necessary steps for the complete determination of a three-dimensional structure by X-ray diffraction techniques. The basic procedures used for protein isolation and crystallization, crystallographic data collection and analysis and, finally, structure determination and refinement are all briefly reviewed. As such our efforts are not directed towards the specialist. Rather, it is our hope that the information presented will aid interested readers from other fields in the understanding of more specialized literature and who may wish to employ the information contained therein in the planning of their biological research. We hope that in so doing we will make clear both the power and limitations of the technique.

Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data.

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.

The allosteric transition of glucosamine-6-phosphate deaminase: the structure of the T state at 2.3 A resolution.

BACKGROUND: The allosteric hexameric enzyme glucosamine-6-phosphate deaminase from Escherichia coli catalyses the regulatory step of N-acetylglucosamine catabolism, which consists of the isomerisation and deamination of glucosamine 6-phosphate (GlcN6P) to form fructose 6-phosphate (Fru6P) and ammonia. The reversibility of the catalysis and its rapid-equilibrium random kinetic mechanism, among other properties, make this enzyme a good model for studying allosteric processes. RESULTS: Here we present the structure of P6(3)22 crystals, obtained in sodium acetate, of GlcN6P deaminase in its ligand-free T state. These crystals are very sensitive to X-ray radiation and have a high (78%) solvent content. The activesite lid (residues 162-185) is highly disordered in the T conformer; this may contribute significantly to the free-energy change of the whole allosteric transition. Comparison of the structure with the crystallographic coordinates of the R conformer (Brookhaven Protein Data Bank entry 1 dea) allows us to describe the geometrical changes associated with the allosteric transition as the movement of two rigid entities within each monomer. The active site, located in a deep cleft between these two rigid entities, presents a more open geometry in the T conformer than in the R conformer. CONCLUSIONS: The differences in active-site geometry are related to alterations in the substrate-binding properties associated with the allosteric transition. The rigid nature of the two mobile structural units of each monomer seems to be essential in order to explain the observed kinetics of the deaminase hexamer. The triggers for both the homotropic and heterotropic allosteric transitions are discussed and particular residues are assigned to these functions. A structural basis for an entropic term in the allosteric transition is an interesting new feature that emerges from this study.

Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase: structure, catalytic mechanism and targeted inhibitor design.

The structure of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from glycosomes of the parasite Trypanosoma cruzi, causative agent of Chagas' disease, is reported. The final model at 2.8 A includes the bound cofactor NAD+ and 90 water molecules per monomer and resulted in an Rfactor of 20.1%, Rfree = 22.3%, with good geometry indicators. The structure has no ions bound at the active site resulting in a large change in the side chain conformation of Arg249 which as a consequence forms a salt bridge to Asp210 in the present structure. We propose that this conformational change could be important for the reaction mechanism and possibly a common feature of many GAPDH structures. Comparison with the human enzyme indicates that interfering with this salt bridge could be a new approach to specific inhibitor design, as the equivalent to Asp210 is a leucine in the mammalian enzymes.

Crystallographic and spectroscopic characterization of a molecular hinge: conformational changes in bothropstoxin I, a dimeric Lys49-phospholipase A2 homologue.

Bothropstoxin I (BthTX-I) from the venom of Bothrops jararacussu is a myotoxic phospholipase A2 (PLA2) homologue which, although catalytically inactive due to an Asp49-->Lys substitution, disrupts the integrity of lipid membranes by a Ca2+-independent mechanism. The crystal structures of two dimeric forms of BthTX-I which diffract X-rays to resolutions of 3.1 and 2.1 angstroms have been determined. The monomers in both structures are related by an almost perfect twofold axis of rotation and the dimer interfaces are defined by contacts between the N-terminal alpha-helical regions and the tips of the beta-wings of partner monomers. Significant differences in the relative orientation of the monomers in the two crystal forms results in "open" and "closed" dimer conformations. Spectroscopic investigations of BthTX-I in solution have correlated these conformational differences with changes in the intrinsic fluorescence emission of the single tryptophan residues located at the dimer interface. The possible relevance of this structural transition in the Ca2+-independent membrane damaging activity is discussed.