ABSTRACT
The crystallization conditions of the synthetic RNA duplex r(GCGGCGU)*r(GCGCCGC), part of the Thermus flavus 5S rRNA domain B, were investigated in detail. The crystallization analysis revealed a relative narrow crystallization zone. Single sequence variations did not enhance the crystal quality, however the crystallization under microgravity provided crystals of higher quality. They belong to the space group P3(1)21 with unit cell dimensions of a = b = 35.0 A and c = 141.2 A. Diffraction data up to 2.6 A were collected and the structure subsequently analysed and refined to an R-value of 22.4 %. The conformation of the two molecules in the asymmetric unit is stabilized by intermolecular hydrogen bonds. The two molecules A and B are perpendicular to each other and interacting head to tail with symmetry related molecules. They form pseudo-continuous infinite helices in the crystal lattice.
Subject(s)
Crystallization/methods , RNA, Bacterial/chemistry , RNA, Ribosomal, 5S/chemistry , Thermus/chemistry , Base Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Molecular Structure , Nucleic Acid Conformation , RNA, Bacterial/genetics , RNA, Ribosomal, 5S/genetics , Thermus/genetics , WeightlessnessABSTRACT
The crystal structure of the ribosome-inactivating protein (RIP) mistletoe lectin I (ML-I) from Viscum album in complex with adenine has been refined to 1.9 A resolution. High quality crystals of the ML-I complex were obtained by the method of vapour diffusion using the high density protein crystal growth system (HDPCG) on the international space station, mission ISS 6A. Hexagonal crystals were grown during three months under microgravity conditions. Diffraction data to 1.9A were collected applying synchrotron radiation and cryo- techniques. The structure was refined subsequently to analyse the structure of ML-I and particularly the active site conformation, complexed by adenine that mimics the RNA substrate binding.
Subject(s)
Crystallization/methods , Plant Preparations/chemistry , Plant Proteins , Toxins, Biological/chemistry , Adenosine Monophosphate , Binding Sites , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Ribosome Inactivating Proteins, Type 2 , Static Electricity , Viscum album/chemistry , WeightlessnessABSTRACT
Fueled by initial space shuttle results, the National Aeronautics and Space Administration (NASA) has been supporting fundamental studies of macromolecular crystal growth since 1985. The majority of this research is directed at understanding the relationship between experimental variables and important crystal characteristics. The program has resulted in new methods and technology that will benefit the crystallography community's effort to meet the ever-increasing demand for protein structural information. Microgravity crystallization results indicate a potential impact on structural biology's more challenging problems, as soon as long-duration experiments can be performed on the International Space Station.
ABSTRACT
The NH(3)-dependent NAD(+) synthetase (NADS) participates in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) by transforming nicotinic acid adenine dinucleotide (NaAD) to NAD(+). The structural behavior of the active site, including stabilization of flexible loops 82-87 and 204-225, has been studied by determination of the crystal structures of complexes of NADS with natural substrates and a substrate analog. Both loops are stabilized independently of NaAD and solely from the ATP-binding site. Analysis of the binding contacts suggests that the minor loop 82-87 is stabilized primarily by a hydrogen bond with the adenine base of ATP. Formation of a coordination complex with Mg(2+) in the ATP-binding site may contribute to the stabilization of the major loop 204-225. The major loop has a role in substrate recognition and stabilization, in addition to the protection of the reaction intermediate described previously. A second and novel Mg(2+) position has been observed closer to the NaAD-binding site in the structure crystallized at pH 7.5, where the enzyme is active. This could therefore be the catalytically active Mg(2+).
Subject(s)
Amide Synthases/chemistry , Bacillus subtilis/enzymology , Amide Synthases/genetics , Amide Synthases/metabolism , Ammonia/chemistry , Binding Sites , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Enzyme Stability , Magnesium/chemistry , Protein Conformation , Substrate SpecificityABSTRACT
Proteinese K (PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess, under microgravity conditions. The intensity data were collected at 4 degrees C to 1.8 A resolution and the final R-factor after refinement for all the reflections was 0.164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 A and 2.58 A respectively from Cys-73 Sgamma. The Cys-73 in the enzyme structure is located close to the active site residue, His-69. This region is completely buried and is not accessible to the solvent. It is rather tightly packed. Therefore, the binding of mercury distorts the stereochemistry of the neighbouring residues including those belonging to the catalytic triad. As a result of this, the Ogamma of Ser-224 is displaced by 0.6 A which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 A between Ser-224 Ogamma and His-69 Nepsilon2.
Subject(s)
Endopeptidase K/chemistry , Fungal Proteins/chemistry , Mercury/pharmacology , Amino Acids/chemistry , Ascomycota/enzymology , Binding Sites/drug effects , Crystallization , Crystallography, X-Ray , Cysteine , Endopeptidase K/metabolism , Fungal Proteins/metabolism , Hydrogen Bonding , Molecular Conformation , Protein Structure, TertiaryABSTRACT
The three-dimensional structure of the ternary complex consisting of human alpha-thrombin, hirugen and the active-site inhibitor RWJ-51438 has been determined at 1.7 A resolution. The crystals of the complex belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 62.98, b = 117.52, c = 47.99 A. The refined R and R(free) values are 0.196 and 0.232, respectively. The ketone carbonyl group of the inhibitor is covalently linked to the hydroxyl O atom of Ser195, forming a tetrahedral intermediate hemiketal structure; the benzothiazole ring N atom of RWJ-51438 forms a hydrogen bond with His57. Surprisingly, the carboxylate substituent on the benzothiazole group forms salt bridges with Lys60F NZ and the NZ of the symmetry-related residues Lys236 and Lys240, which introduces steric effects that perturb the 60A-60I insertion loop, especially at residues Trp60D and Phe60H.
Subject(s)
Antithrombins/chemistry , Oligopeptides/chemistry , Thiazoles/chemistry , Thrombin/chemistry , Benzothiazoles , Binding Sites , Humans , Models, Molecular , Protein ConformationABSTRACT
Dengue viruses are members of the Flaviviridae and cause dengue fever and the more severe dengue hemorrhagic fever. Although nearly 40 % of the world's population is at risk of dengue infection, there is currently no effective vaccine or chemotherapy for the disease. Processing of the dengue polyprotein into structural and non-structural proteins in a host, which is essential for assembly of infective virions, is carried out by the combined action of host proteases and the trypsin-like, two-component viral NS2B/NS3 serine protease. Although NS2B strongly stimulates the catalytic NS3 protease domain, the latter is fully active against small substrates and possesses detectable activity against larger substrates, making both forms of the enzyme possible targets for drug design. In the crystal structure of a complex of the protease with a Bowman-Birk inhibitor reported here, an Arg residue at the P1 position of the inhibitor is bound in a manner distinctly different from that in other serine proteases of comparable specificity. However, because the regulatory component, NS2B, is not present in the complex, the physiological implications of this observations are currently unclear. The redundant nature of interaction of P1 Arg and Lys residues with Asp129, Tyr150 and Ser163 of the enzyme provides an explanation for the observed behavior of several site-specific mutants of Asp129 in the protease. The strong level of conservation of residues in the protease that interact with the P1 Arg, along with conservation of Arg at P1 of most cleavage sites in other flaviviruses, suggests that observations from this structure are likely to be applicable to many flaviviruses. The structure provides a starting point for design of site-specific mutations to probe the mechanism of catalysis by the catalytic domain, its activation by the regulatory domain and for design of specific inhibitors of enzymatic activity.
Subject(s)
Dengue Virus/enzymology , Drug Design , Protein Processing, Post-Translational , Trypsin Inhibitor, Bowman-Birk Soybean/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Catalytic Domain , Crystallography, X-Ray , Dengue Virus/genetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Ligands , Models, Molecular , Mutation , Polyproteins/chemistry , Polyproteins/metabolism , Protein Conformation , RNA Helicases , Serine Endopeptidases , Substrate Specificity , Trypsin Inhibitor, Bowman-Birk Soybean/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Rab proteins are small Ras-like GTPases which play important roles in regulating intracellular vesicle trafficking. The nucleotide-binding domain of Rab6 from the malaria parasite Plasmodium falciparum was crystallized with GDP bound to the active site. The MAD phasing technique was used to determine the crystal structure to 2.3 A resolution. Comparisons of the structure of GDP-bound PfRab6 with the recently determined structures of Rab3A in complex with either a GTP analog or with GTP and Rabphillin present structural evidence supporting the traditional model for the molecular GTP/GDP switch in Rab proteins. PfRab6 residues homologous to those distinguishing human Rab6 isoforms, which differ in binding to Rabkinesin-6 in human cells, are located next to the recognized complementarity-determining region (CDR) and constitute a conceptual broadening of that domain. Despite significant observable differences in Golgi ultrastructure, the Rab6 core structure and switch mechanism appear highly conserved when compared with murine Rab3a structures. A significant difference between the PfRab6 and higher eukaryotic Rabs may be the lack of CDR features that allow binding interactions with Rabkinesin-type effectors.
Subject(s)
Plasmodium falciparum/chemistry , Protozoan Proteins/chemistry , rab GTP-Binding Proteins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Protein Conformation , Protein Structure, Tertiary , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Sequence Homology, Amino Acid , Static Electricity , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Selective transport of polymeric (p) immunoglobulins (Ig) of IgA and IgM isotypes into external secretions by pIg receptor-mediated mechanism depends on the incorporation of joining (J) chain into the polymers. Until now, availability of a free J chain for immunological and biophysical studies has been limited to preparations of denatured J chain forms with moderate yield. Here we report that a recombinant J chain (rJ) can be over-expressed as a soluble fusion protein with thioredoxin using a modified vector pET32 in Escherichia coli. An intact J chain was released by digestion with IgA1 protease from Neisseria gonorrhoeae and isolated in a good yield with immunological and biochemical properties similar to those of J chain obtained by chemical cleavage from pIgA.
Subject(s)
Immunoglobulin J-Chains/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Humans , Immunoglobulin A , Immunoglobulin J-Chains/genetics , Immunoglobulin M , Molecular Sequence Data , Serine Endopeptidases/metabolism , Thioredoxins/biosynthesis , Thioredoxins/geneticsABSTRACT
Human infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 A resolution to an R-factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five-residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.
Subject(s)
Adenosine Kinase/chemistry , Toxoplasma/enzymology , Adenosine/metabolism , Adenosine Kinase/metabolism , Amino Acid Sequence , Animals , Binding Sites , Chlorides/metabolism , Crystallography, X-Ray , Humans , Magnesium/metabolism , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Thermus flavus 5S rRNA with a molecular weight of about 40 kDa was modified at the 5' and 3' ends. Crystals were obtained under earth and microgravity conditions. The best crystals were obtained during NASA space mission STS 94. For the first time, it was possible to collect a complete data set from 5S rRNA crystals to 7.8 A resolution and to assign the space group as R32, with unit-cell parameters a = b = 110.3, c = 387.6 A, alpha = beta = 90, gamma = 120 degrees.
Subject(s)
Nucleic Acid Conformation , RNA, Ribosomal, 5S/chemistry , Thermus/chemistry , Base Sequence , Crystallization , Crystallography, X-Ray , Genetic Engineering , Gravitation , Models, Molecular , Molecular Sequence Data , Molecular Weight , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , RNA, Ribosomal, 5S/genetics , RNA, Ribosomal, 5S/isolation & purification , Thermus/genetics , WeightlessnessABSTRACT
The obligate intracellular protozoan parasite Toxoplasma gondii depends on the purine-salvage pathway for its purine supply. Unlike its mammalian hosts, T. gondii salvages purine precursors predominantly via adenosine kinase, the enzyme that phosphorylates adenosine to adenosine monophosphate (AMP). The cDNA encoding T. gondii adenosine kinase was subcloned and expressed in Escherichia coli. The recombinant protein was active in an in vitro enzyme assay over a broad pH range. It required a divalent cation for activity. The enzyme was inactivated by the addition of 1 microM mercuric chloride. The inactivation could be reversed by a reducing agent. The active recombinant protein was crystallized using sodium sulfate as precipitant at pH 8.0. The crystals diffract to 1.8 A and belong to the monoclinic space group P2(1), with unit-cell parameters a = 47.5, b = 68.9, c = 57.0 A, beta = 100.3 degrees. The calculated V(m) based on one molecule per asymmetric unit is 2.38 A(3) Da(-1).
Subject(s)
Adenosine Kinase/chemistry , Adenosine Kinase/isolation & purification , Toxoplasma/enzymology , Adenosine Kinase/genetics , Animals , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Escherichia coli/genetics , Gene Expression , Genes, Protozoan , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Toxoplasma/geneticsABSTRACT
Escherichia coli NAD synthetase was overexpressed and purified to homogeneity. The recombinant protein was active in an in vitro enzyme assay. The enzyme required approximately 1.5 mM magnesium for optimal activity. The pH optimum was found to be 8.0-8.5. The recombinant protein was crystallized at room temperature using the hanging-drop vapor diffusion technique with 1.5 M lithium sulfate, 0. 1 M Hepes buffer at pH 7.5 as precipitant. The protein was also crystallized in the presence of its substrates, nicotinic acid adenine dinucleotide and adenosine triphosphate under similar conditions. These crystals diffract to 2.0-A resolution and belong to trigonal space group P3(1)21 with unit cell dimensions of a = b = 91.766, c = 74.17 A and alpha = beta = 90 degrees, gamma = 120 degrees. The structure of the complex has been determined using the molecular replacement method.
Subject(s)
Amide Synthases/chemistry , Escherichia coli/enzymology , Amide Synthases/genetics , Amide Synthases/isolation & purification , Amino Acid Sequence , Cloning, Molecular , Computer Graphics , Crystallization , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
The three-dimensional structure of bovine pancreatic trypsin complexed with the inhibitor RWJ-51084 has been determined at 1.8 A resolution. These crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 53.43, c = 107.76 A. The refined R and R(free) values are 0.175 and 0.237, respectively. The carbonyl group bonded to the benzothiazole group of the inhibitor is covalently linked to the hydroxyl O atom of Ser195, forming a tetrahedral intermediate hemiketal structure. The other carbonyl O atom of the inhibitor forms a hydrogen bond with the Gln192 side-chain amide group. The benzothiazole group is oriented with the aromatic N atom of RWJ-51084 accepting a hydrogen bond from His57 NE2. The arginine side chain of the inhibitor extends into the deep and narrow pocket of the S1 specificity site of trypsin, forming a network of hydrogen bonds.
Subject(s)
Arginine/analogs & derivatives , Serine Proteinase Inhibitors/chemistry , Thiazoles/chemistry , Trypsin/chemistry , Animals , Arginine/chemistry , Benzothiazoles , Binding Sites , Cattle , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Molecular Structure , Pancreas/enzymology , Protein Folding , Protein Structure, Secondary , Thrombin/chemistry , Trypsin Inhibitors/chemistryABSTRACT
PURPOSE: The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. The purpose of this study is twofold: (1) to compare the activity of N-acetyl-PGP with the bioactivities of other similar synthetic peptides in an effort to uncover information about this chemoattractant molecule, and (2) to test these peptide analogs as potential antagonists of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of human neutrophils to peptides. Bioactivity was expressed as the peptide concentration required to produce 50% neutrophil polarization (EC50). Antagonist activity was expressed as the peptide concentration required to produce 50% inhibition (ID50) of polarization activated by N-acetyl-PGP. RESULTS: Peptide bioactivities (EC50) were ranked as follows: APGPR (0.34 mM) > N-acetyl-PGP (0.5 mM) > N-(PGP)4-PGLG (3 mM) = t-Boc-PGP (3 mM) > N-acetyl-PG (3.4 mM) > N-methyl-PGP (15 mM) = PGP (15 mM) > peptides without detectable activity (t-Boc-PGP-OMe, N-acetyl-P, PG, PGG, GP, GG and gly-pro-hyp). Peptides with no detectable bioactivity were tested as potential antagonists of neutrophil polarization induced by N-acetyl-PGP. Gly-Pro-Hyp inhibited N-acetyl-PGP activation of polarization at 20 mM (ID50). No other synthetic peptide demonstrated a capacity for inhibition. CONCLUSIONS: The minimum requirement to elicit bioactivity was the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. Using this approach, active and inactive mimetic peptides of N-acetyl-PGP were produced. The most active peptide, APGPR, was equal to or slightly greater than N-acetyl-PGP, suggesting that more potent analogs might be designed. Gly-pro-hyp was the only inactive peptide analog to inhibit the chemoattractant.
Subject(s)
Chemotactic Factors/pharmacology , Chemotaxis, Leukocyte/physiology , Neutrophil Activation/drug effects , Neutrophils/physiology , Oligopeptides/pharmacology , Proline/analogs & derivatives , Chemotactic Factors/chemical synthesis , Humans , Oligopeptides/chemical synthesis , Proline/chemical synthesis , Proline/pharmacology , Structure-Activity RelationshipABSTRACT
Crystallization and preliminary characterization of the essential dengue virus NS3 serine protease complexed with a Bowman-Birk-type inhibitor from mung beans are reported. As the structure proved resistant to solution by molecular replacement and multiple isomorphous replacement methods, multi-wavelength anomalous diffraction data at the LIII edge of a holmium derivative have been measured. Promising Bijvoet and dispersive signals which are largely consistent with expected values have been extracted from the data. The structure, when determined, will provide a structural basis for the design, synthesis and evaluation of inhibitors of the protease for chemotherapy of dengue infections.
Subject(s)
Dengue Virus/enzymology , Trypsin Inhibitor, Bowman-Birk Soybean/chemistry , Viral Nonstructural Proteins/chemistry , Crystallization , Crystallography, X-Ray , Protein Conformation , RNA Helicases , Serine EndopeptidasesABSTRACT
Recombinant proteins of monomeric and dimeric B-domain repeats of a Staphylococcus aureus FDA 574 collagen-binding adhesin have been crystallized. The single repeat unit (B1) was crystallized in a body-centered orthorhombic lattice with a = 96.9, b = 101.3, c = 120. 8 A in either the I222 or I212121 space group. These crystals diffracted to 2.5 A resolution and the calculated Vm values of 3.2 and 2.2 A3 Da-1 suggest the possibility of a dimer or a trimer in the asymmetric unit. The two-repeat fragment (B1B2) crystallized in the orthorhombic space group P212121 with cell dimensions a = 42.4, b = 79.4, c = 130.4 A and diffracted to 2.3 A resolution. For this species, the calculated Vm value of 2.2 A3 Da-1 indicates the presence of a monomer in the asymmetric unit.