Preliminary crystallographic studies of a Schistosoma mansoni antigen (Sm21.7) dynein light-chain (DLC) domain.

Schistosomiasis is an inflammatory chronic disease that represents a major health problem in tropical and subtropical countries. The drug of choice for treatment, praziquantel, is effective in killing adult worms but fails to kill immature forms and prevent reinfection. One prominent antigen candidate for an anti-schistosomiasis vaccine is the protein Sm21.7 (184 amino-acid residues) from Schistosoma mansoni, a tegumental protein capable of reducing the worm burden in a murine immunization model. In the present work, the Sm21.7 gene was cloned and expressed in Escherichia coli and the full-length protein was purified to homogeneity. Crystals of recombinant Sm21.7 suitable for X-ray diffraction were obtained using PEG monomethyl ether 2000 as a precipitant. X-ray diffraction images of a native crystal (at 2.05 Šresolution) and a quick-cryosoaked NaI derivative (at 1.95 Šresolution) were collected on the W01B-MX2 beamline at the Laboratório Nacional de Luz Síncrotron (LNLS, Brazilian Synchrotron Light Laboratory/MCT). Both crystals belonged to the hexagonal space group P6122, with similar unit-cell parameters a=b=108.5, c=55.8 Å. SIRAS-derived phases were used to generate the first electron-density map, from which a partial three-dimensional model of Sm21.7 (from Gln89 to Asn184) was automatically constructed. Anaysis of dissolved crystals by SDS-PAGE confirmed that the protein was cleaved in the crystallization drop and only the Sm21.7 C-terminal domain was crystallized. The structure of the Sm21.7 C-terminal domain will help in the localization of the epitopes responsible for its protective immune responses, constituting important progress in the development of an anti-schistosomiasis vaccine.

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 structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition.

Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked beta-d-fructofuranose residues in inulin, levan and sucrose releasing beta-d-fructose. We present the X-ray structure at 1.55A resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed beta-propeller fold and the C-terminal domain folded into a beta-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (beta-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87A, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.

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 alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

Crystallization and synchrotron X-ray diffraction studies of human interleukin-22.

Human interleukin-22, a novel member of the cytokine family, has been crystallized in hanging drops using the vapour-diffusion technique. Preliminary X-ray diffraction experiments using synchrotron radiation reveal that the protein crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 55.44, b = 61.62, c = 73.43 A, and diffracts beyond 2.00 A resolution.

Crystallization and preliminary X-ray diffraction analysis of a novel trypsin inhibitor from seeds of Copaifera langsdorffii.

A novel trypsin inhibitor isolated from seeds of Copaifera langsdorffii was purified to homogeneity and crystallized. Crystals suitable for X-ray analysis were grown using the hanging-drop vapour-diffusion method at 291 K in sodium acetate buffer at pH values near 4.3 using PEG 4000 as precipitant. The crystals presented symmetry compatible with the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 58.71, c = 93.75 A, and diffracted to 1.83 A resolution at the synchrotron source.

Protein crystal structure solution by fast incorporation of negatively and positively charged anomalous scatterers.

The preparation of derivatives by the traditional methods of soaking is one of the most time-consuming steps in protein crystal structure solution by X-ray diffraction techniques. The 'quick cryosoaking' procedure for derivatization with halides (monovalent anions) offers the possibility of significantly speeding up this process [Dauter et al. (2000), Acta Cryst. D56, 232-237]. In the present work, an extension of this technique is proposed and the use of two different classes of compounds (monovalent and polyvalent cations) that can be successfully utilized in the quick cryosoaking procedure for the derivatization and phasing of protein crystals is described. This approach has been tested on hen egg-white lysozyme and has been successfully used to solve the structure of a novel trypsin inhibitor. The possibility of using cations in the fast cryosoaking procedure gives additional flexibility in the process of derivatization and increases the chances of success in phase determination. This method can be applied to high-throughput crystallographic projects.

Crystallization and preliminary X-ray study of haem-binding protein from the bloodsucking insect Rhodnius prolixus.

Rhodnius haem-binding protein (RHBP) from the bloodsucking insect Rhodnius prolixus, a 15 kDa protein, has been crystallized using polyethylene glycol as a precipitant. X-ray diffraction data have been collected at a synchrotron source. The crystals belong to the space group P4(1(3))2(1)2, with unit-cell parameters a = b = 64.98, c = 210.68 A, and diffract beyond 2.6 A resolution.

Crystallization and preliminary X-ray diffraction studies of human catalase.

The enzyme catalase (H(2)O(2)-H(2)O(2) oxidoredutase; E.C. 11.1.6) was purified from haemolysate of human placenta and crystallized using the vapour-diffusion technique. Synchrotron-radiation diffraction data have been collected to 1.76 A resolution. The enzyme crystallized in the space group P2(1)2(1)2(1), with unit-cell dimensions a = 83.6, b = 139.4, c = 227.5 A. A molecular-replacement solution of the structure has been obtained using beef liver catalase (PDB code 4blc) as a search model.