ABSTRACT
Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.
Subject(s)
Coenzymes/chemistry , Crystallization , IMP Dehydrogenase/chemistry , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Animals , Binding Sites , Catalytic Domain , Cloning, Molecular , Guanosine Monophosphate , Models, Molecular , Protein Conformation , Sf9 Cells , Trypanosoma brucei brucei/geneticsABSTRACT
During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9â Å resolution using automatic model-building tools.
ABSTRACT
Pyrimidine-nucleoside phosphorylase catalyzes the phosphorolytic cleavage of thymidine and uridine with equal activity. Investigation of this protein is essential for anticancer drug design. Here, the structure of this protein from Bacillus subtilis in complex with imidazole and sulfate is reported at 1.9â Å resolution, which is an improvement on the previously reported structure at 2.6â Å resolution. The localization and position of imidazole in the nucleoside-binding site reflects the possible binding of ligands that possess an imidazole ring.
Subject(s)
Bacillus subtilis/enzymology , Imidazoles/metabolism , Pyrimidine Phosphorylases/chemistry , Pyrimidine Phosphorylases/metabolism , Sulfates/metabolism , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallization , Crystallography, X-Ray , Imidazoles/chemistry , Models, Molecular , Protein Conformation , Substrate Specificity , Sulfates/chemistryABSTRACT
Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.
ABSTRACT
X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus µNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitro.
Subject(s)
Toxicology , Toxicology , Poisons , Poisoning , Toxins, Biological , Toxicology , BiochemistryABSTRACT
Catalytically inactive phospholipase A(2) (PLA(2)) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA(2) homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA(2) homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA(2) homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement.
Subject(s)
Mutant Proteins/chemistry , Phospholipases A2/chemistry , Animals , Catalytic Domain , Ethylene Glycols/chemistry , Ligands , Models, Molecular , Mutant Proteins/isolation & purification , Phospholipases A2/isolation & purification , Protein Conformation , Protein Multimerization , Protein Structure, SecondaryABSTRACT
L-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate L-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH(3)) and hydrogen peroxide (H(2)O(2)). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1 Å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode.
Subject(s)
Bothrops , Crotalid Venoms/enzymology , L-Amino Acid Oxidase/chemistry , Amino Acid Sequence , Animals , Crystallography, X-Ray , Enzyme Stability , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Protein Structure, SecondaryABSTRACT
The venom of Antarctic octopus remains completely unstudied. Here, a preliminary investigation was conducted into the properties of posterior salivary gland (PSG) extracts from four Antarctica eledonine (Incirrata; Octopodidae) species (Adelieledone polymorpha, Megaleledone setebos, Pareledone aequipapillae, and Pareledone turqueti) collected from the coast off George V's Land, Antarctica. Specimens were assayed for alkaline phosphatase (ALP), acetylcholinesterase (AChE), proteolytic, phospholipase A(2) (PLA(2)), and haemolytic activities. For comparison, stomach tissue from Cirroctopus sp. (Cirrata; Cirroctopodidae) was also assayed for ALP, AChE, proteolytic and haemolytic activities. Dietary and morphological data were collected from the literature to explore the ecological importance of venom, taking an adaptive evolutionary approach. Of the incirrate species, three showed activities in all assays, while P. turqueti did not exhibit any haemolytic activity. There was evidence for cold-adaptation of ALP in all incirrates, while proteolytic activity in all except P. turqueti. Cirroctopus sp. stomach tissue extract showed ALP, AChE and some proteolytic activity. It was concluded that the AChE activity seen in the PSG extracts was possibly due to a release of household proteins, and not one of the secreted salivary toxins. Although venom undoubtedly plays an important part in prey capture and processing by Antarctica eledonines, no obvious adaptations to differences in diet or morphology were apparent from the enzymatic and haemolytic assays. However, several morphological features including enlarged PSG, small buccal mass, and small beak suggest such adaptations are present. Future studies should be conducted on several levels: Venomic, providing more detailed information on the venom compositions as well as the venom components themselves; ecological, for example application of serological or genetic methods in identifying stomach contents; and behavioural, including observations on capture of different types of prey.
Subject(s)
Adaptation, Physiological , Cold Temperature , Environment , Mollusk Venoms/analysis , Octopodiformes/physiology , Alkaline Phosphatase/metabolism , Animals , Antarctic Regions , Cholinesterase Inhibitors/metabolism , Erythrocytes/drug effects , Female , Hemolysis , Male , Mollusk Venoms/enzymology , Mollusk Venoms/pharmacology , Octopodiformes/anatomy & histology , Octopodiformes/classification , Phenotype , Phylogeny , Salivary Glands/chemistry , Salivary Glands/metabolismABSTRACT
The mammalian peptidoglycan recognition protein-S (PGRP-S) binds to peptidoglycans (PGNs), which are essential components of the cell wall of bacteria. The protein was isolated from the samples of milk obtained from camels with mastitis and purified to homogeneity and crystallized. The crystals belong to orthorhombic space group I222 with a=87.0 A, b=101.7 A and c=162.3 A having four crystallographically independent molecules in the asymmetric unit. The structure has been determined using X-ray crystallographic data and refined to 1.8 A resolution. Overall, the structures of all the four crystallographically independent molecules are identical. The folding of PGRP-S consists of a central beta-sheet with five beta-strands, four parallel and one antiparallel, and three alpha-helices. This protein fold provides two functional sites. The first of these is the PGN-binding site, located on the groove that opens on the surface in the direction opposite to the location of the N terminus. The second site is implicated to be involved in the binding of non-PGN molecules, it also includes putative N-terminal segment residues (1-31) and helix alpha2 in the extended binding. The structure reveals a novel arrangement of PGRP-S molecules in which two pairs of molecules associate to form two independent dimers. The first dimer is formed by two molecules with N-terminal segments at the interface in which non-PGN binding sites are buried completely, whereas the PGN-binding sites of two participating molecules are fully exposed at the opposite ends of the dimer. In the second dimer, PGN-binding sites are buried at the interface while non-PGN binding sites are fully exposed at the opposite ends of the dimer. This form of dimeric arrangement is unique and seems to be aimed at enhancing the capability of the protein against specific invading bacteria. This mode of functional dimerization enhances efficiency and specificity, and is observed for the first time in the family of PGRP molecules.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Carrier Proteins/genetics , Crystallography, X-Ray , Dimerization , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The electrophile Ca(2+) is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca(2+) free and bound states at 0.97 and 1.60 A resolutions, respectively. In the Ca(2+) bound state, the Ca(2+) ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca(2+), a water molecule occupies the position of the Ca(2+) ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation.
Subject(s)
Calcium/metabolism , Phospholipases A/chemistry , Phospholipases A/metabolism , Animals , Binding Sites , Bothrops/metabolism , Crotalid Venoms/enzymology , Crystallization , Crystallography, X-Ray/methods , Group IV Phospholipases A2 , Hydrogen Bonding , Models, Molecular , Phospholipases A2 , Protein Binding , Protein Structure, Secondary , Protein Structure, TertiarySubject(s)
Toxicology , Toxicology , Poisons , Poisoning , Toxins, Biological , Toxicology , BiochemistryABSTRACT
Ribonuclease T1 is an enzyme that cleaves single-stranded RNA with high specificity after guanylyl residues. Although this enzyme is a very good characterized protein with respect to structure and enzymatic function, we were only recently successful in generating RNase T1-RV, a variant where the specificity was changed from guanine to purine. As this change of substrate specificity was made at the cost of activity, the aim was now to further improve the overall activity of the enzyme. Therefore, we have substituted the tryptophan in position 59 by tyrosine. This substitution led to an increase of enzymatic activity in comparison to variant RV to 425%. As the extent of this enhancement is unique so far we have crystallized and analyzed the structure of this variant in order to get more insights into the reasons for this. Here, we present the crystal structure of this so-called RNase T1-R2 at 2.1A resolution. The structure was determined by molecular replacement using the coordinates of the RV variant (PDB entry: 1Q9E). The data were refined to an R-factor of 18.7% and R(free) of 24%, respectively. The asymmetric unit contains three molecules and the crystal packing is very similar to that of variant RV.
Subject(s)
Purines/metabolism , Ribonuclease T1/chemistry , Ribonuclease T1/genetics , Tyrosine/chemistry , Amino Acid Substitution , Catalytic Domain , Kinetics , Models, Molecular , Ribonuclease T1/metabolism , Substrate Specificity , Tryptophan/chemistry , Tryptophan/genetics , Tyrosine/geneticsABSTRACT
An easy method to crystallize homogenous HAP at physiological pH as well as powders of HAP and CPP at low temperature are described. Platy and spherulitic crystals of HAP were crystallized at the physiological pH using single diffusion method. Well-defined platy crystals of hydroxyapatite were obtained at the physiological temperature and pH. These crystals were found to be pure and homogenous form of HAP without any contamination from the crystallizing medium. Spherulitic crystals of HAP of approximately 3 mm in diameter were obtained in the presence of Fe at 47 degrees C. A sol-gel technique involving agarose is described for the preparation of hydroxyapatite and calcium pyrophosphate. Pure form of HAP was synthesised at 85 degrees C and its sintering properties were also studied. At a temperature of 1200 degrees C, the material gets completely converted to alpha-calcium pyrophosphate. The samples were analysed by XRD, IR, TGA and SEM. The particle size of the synthesised powders was measured using the dynamic light scattering experiments.
Subject(s)
Biocompatible Materials/chemistry , Crystallization/methods , Durapatite/chemistry , Temperature , Hydrogen-Ion Concentration , Materials Testing , Molecular Conformation , Particle Size , Powders , Surface PropertiesABSTRACT
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
Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62A resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the pi electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-alpha-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same.
Subject(s)
Antigens, Tumor-Associated, Carbohydrate/chemistry , Antigens, Tumor-Associated, Carbohydrate/metabolism , Artocarpus/chemistry , Lectins/chemistry , Lectins/metabolism , Plant Lectins/chemistry , Plant Lectins/metabolism , Binding Sites , Carbohydrate Conformation , Crystallization , Crystallography, X-Ray , Galactose/metabolism , Hydrogen Bonding , Models, Molecular , Oxygen/metabolism , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits , Substrate SpecificityABSTRACT
Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water-magnesium complex involved in their catalytic acts. In the enzyme-substrate complexes, the Mg2+ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3'-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3'-leaving group. A strained metalloenzyme-substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction. The English version of the paper.
Subject(s)
Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Physarum polycephalum/enzymology , Serratia marcescens/enzymology , Amino Acid Sequence , Animals , Crystallography, X-Ray , Magnesium , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
The crystal structure of phospholipase A(2) from the venom of Daboia russelli pulchella has been refined to an R factor of 0.216 using 17,922 reflections to 1.9 A resolution. The structure contains two crystallographically independent molecules in the asymmetric unit. The overall conformations of the two molecules are essentially the same except for three regions, namely the calcium-binding loop including Trp31, the beta-wing and the C-terminal residues 119-131. Although these differences have apparently been caused by molecular packing, they seem to have functional relevance. Particularly noteworthy is the conformation of Trp31, which is favourable for substrate binding in one molecule as it is aligned with one of the side walls of the hydrophobic channel, whereas in the other molecule it is located at the mouth of the channel, thereby blocking the entry of substrates leading to loss of activity. This feature is unique to the present structure and does not occur in the dimers and trimers of other PLA(2)s.
Subject(s)
Phospholipases A/chemistry , Viper Venoms/enzymology , Animals , Calcium/metabolism , Catalysis , Crystallization , Crystallography, X-Ray , Models, Molecular , Phospholipases A/isolation & purification , Phospholipases A/metabolism , Phospholipases A2 , Protein Conformation , Protein Structure, Tertiary , Quality Control , ViperidaeABSTRACT
Vipoxin is a neurotoxic postsynaptic heterodimeric complex from the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It consists of a basic and highly toxic phospholipase A(2) and an acidic non-toxic protein inhibitor. The two polypeptide chains have the same chain length and share 62% amino-acid identity. Vipoxin is a unique example of evolution of the catalytic and toxic phospholipase A(2) functions into inhibitory and non-toxic functions. The crystal structure of the complex has been determined by the molecular-replacement method and refined to 1.4 A resolution to an R factor of 18.2%. The complex formation decreases the accessible surface area of the two subunits by approximately 1480 A(2), which results in a reduction of toxicity and catalytic activity. The catalytic and substrate-binding sites of the vipoxin phospholipase A(2) are identical or similar to those of other group I/II enzymes. Two 2-methyl-2,4-pentanediol molecules are present in the hydrophobic channel close to the active site. The two subunits lack calcium ions. The negatively charged Asp49 of the phospholipase A(2), which participates in the Ca(2+)-binding sites of other snake-venom phospholipase A(2)s, is neutralized by the side chain of Lys69 from the inhibitor. Attempts have been made to identify the toxicity region and to explain the reduced catalytic activity and toxicity of the phospholipase A(2) subunit.
Subject(s)
Viper Venoms/chemistry , Amino Acid Sequence , Animals , Binding Sites , Calcium/metabolism , Crystallization , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Phospholipases A/chemistry , Protein Conformation , Sequence Homology, Amino Acid , Viper Venoms/antagonists & inhibitors , ViperidaeABSTRACT
The 'external' oxygenated functional unit RtH2-e of the Rapana hemocyanin subunit RHSS2 was isolated and crystallized. X-ray intensity data to 3.3 A resolution have been collected at 100 K and the structure has been solved using the molecular-replacement method. The space group is assigned to be the tetragonal P4(3)2(1)2, with unit-cell parameters a = b = 105.5, c = 375.0 A.