The crystal structure of a Thermus thermophilus tRNA(Gly) acceptor stem microhelix at 1.6 Å resolution.

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem. The tRNA(Gly)/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA(Gly) acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA(Gly) aminoacyl stem from Thermus thermophilus at 1.6Å resolution and provide insight into the RNA geometry and hydration.

Comparative X-ray structure analysis of human and Escherichia coli tRNA(Gly) acceptor stem microhelices.

tRNA identity elements assure the correct aminoacylation of tRNAs by the aminoacyl-tRNA synthetases with the cognate amino acid. The tRNA(Gly)/glycyl-tRNA sythetase system is member of the so-called 'class II system' in which the tRNA determinants consist of rather simple elements. These are mostly located in the tRNA acceptor stem and in the glycine case additionally the discriminator base at position 73 is required. Within the glycine-tRNA synthetases, the archaebacterial/human and the eubacterial sytems differ with respect to their protein structures and the required tRNA identity elements, suggesting a unique evolutionary divergence. In this study, we present a comparison between the crystal structures of the eubacterial Escherichia coli and the human tRNA(Gly) acceptor stem microhelices and their surrounding hydration patterns.

Crystal structure of a human tRNA(Gly) microhelix at 1.2A resolution.

The tRNA(Gly)/glycyl-tRNA synthetase (GlyRS) system belongs to the so-called 'class II aminoacyl-tRNA synthetase system' in which tRNA identity elements are assured by rather few and simple determinants mostly located in the tRNA acceptor stem. Regarding evolutionary aspects, the tRNA(Gly)/GlyRS system is a special case. There exist two different types of GlyRS, namely an archaebacterial/human type and a eubacterial type reflecting an evolutionary divergence within this system. Here we report the crystal structure of a human tRNA(Gly) acceptor stem microhelix at 1.2A resolution. The local geometric parameters of the microhelix and the water network surrounding the RNA are presented. The structure complements the previously published Escherichia coli tRNA(Gly) aminoacyl stem structure.

High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif.

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.

The mistletoe lectin I--phloretamide structure reveals a new function of plant lectins.

The X-ray structure at 2.7A resolution of the complex between the European mistletoe lectin I (Viscum album, ML-I) and the plant growth hormone, 3-(p-hydroxyphenyl)-propionic acid amide (phloretamide, PA) from xylem sap has revealed the binding of PA at the so far undescribed hydrophobic cavity located between the two subunits of this ribosome-inhibiting protein. No such cavity is observed in related lectins. The binding of PA is achieved through interactions with the non-conserved residues Val228A, Leu230A, Arg388B, and the C-terminal Pro510B. It is conceivable that binding of PA to ML-I is part of a defence mechanism of the parasite against the host, whereby the parasite prevents the growth hormone of the host from interfering with its own regulatory system. The specific binding of PA to ML-I indicates that heterodimeric RIPs are multifunctional proteins whose functions in the cell have not yet been fully recognized and analyzed.

Crystal structure of a calcium-induced dimer of two isoforms of cobra phospholipase A2 at 1.6 A resolution.

The calcium-induced formation of a complex between two isoforms of cobra venom phospholipase A2 reveals a novel interplay between the monomer-dimer and activity-inactivity transitions. The monodispersed isoforms lack activity in the absence of calcium ions while both molecules gain activity in the presence of calcium ions. At concentrations higher than 10 mg/ml, in the presence of calcium ions, they dimerize and lose activity again. The present study reports the crystal structure of a calcium-induced dimer between two isoforms of cobra phospholipase A2. In the complex, one molecule contains a calcium ion in the calcium binding loop while the second molecule does not possess an intramolecular calcium ion. However, there are two calcium ions per dimer in the structure. The second calcium ion is present at an intermolecular site and that is presumably responsible for the dimerization. The calcium binding loops of the two molecules adopt strikingly different conformations. The so-called calcium binding loop in the calcium-containing molecule adopts a normal conformation as generally observed in other calcium containing phospholipase A(2) enzymes while the conformation of the corresponding loop in the calcium free monomer deviates considerably with the formation of a unique intraloop Gly33 (N)-Cys27 (O) = 2.74 A backbone hydrogen bond. The interactions of Arg31 (B) with Asp49 (A) and absence of calcium ion are responsible for the loss of catalytic activity in molecule A while interactions of Arg2 (B) with Tyr52 (B) inactivate molecule B.

First structural evidence of a specific inhibition of phospholipase A2 by alpha-tocopherol (vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A2 and alpha-tocopherol at 1.8 A resolution.

This is the first structural evidence of alpha-tocopherol (alpha-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, alpha-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of alpha-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex.

Sequence-induced trimerization of phospholipase A2: structure of a trimeric isoform of PLA2 from common krait (Bungarus caeruleus) at 2.5 A resolution.

The venom of the common Indian krait (Bungarus caeruleus) contains about a dozen isoforms of phospholipase A2 (PLA2), which exist in different oligomeric forms as well as in complexes with low-molecular-weight ligands. The basic objective of multimerization and complexation is either to inactivate PLA2 in the venom for long-term storage, to generate a new PLA2 function or to make a more lethal assembly. The current isoform was isolated from the venom of B. caeruleus. Dynamic light-scattering studies indicated the presence of a stable trimeric association of this PLA2. Its primary sequence was determined by cDNA cloning. The purified protein was crystallized with 2.8 M NaCl as a precipitating agent using the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 80.9, b = 80.5, c = 57.1 A, beta = 90.3 degrees. The structure was refined to a final R factor of 0.198. This is a novel trimeric PLA2 structure in which the central pore formed by the association of three molecules is filled with water molecules. The interactions across the pore take place via multiple water bridges primarily to the side chains of Arg, Lys and Thr residues. Approximately 12% of the total solvent-accessible surface area is buried in the core of the trimer. The active sites of all three molecules are located on the surface and are fully exposed to the solvent, resulting in a highly potent enzymatic unit.

[X-ray structural analysis of magnesium-containing Serratia marcescens endonuclease]. / Rentgenostrukturnyi analiz magniisoderzhashchei éndonukleazy Serratia marcescens.

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescens was refined at the resolution of 1.07 A to R factor of 12.4% and Rfree factor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg2+ ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N- and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg2+ ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O delta 1 atom of Asn119 were shown to be within a range of 2.01-2.11 A. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 A2, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 A2, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.

Crystal structure of an Escherichia coli tRNA(Gly) microhelix at 2.0 A resolution.

tRNA identity elements determine the correct aminoacylation by the cognate aminoacyl-tRNA synthetase. In class II aminoacyl tRNA synthetase systems, tRNA specificity is assured by rather few and simple recognition elements, mostly located in the acceptor stem of the tRNA. Here we present the crystal structure of an Escherichia coli tRNA(Gly) aminoacyl stem microhelix at 2.0 A resolution. The tRNA(Gly) microhelix crystallizes in the space group P3(2)21 with the cell constants a=b=35.35 A, c=130.82 A, gamma=120 degrees . The helical parameters, solvent molecules and a potential magnesium binding site are discussed.

Superposition of a tRNASer acceptor stem microhelix into the seryl-tRNA synthetase complex.

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNAs. Seryl-tRNA synthetase is a class II synthetase, which depends on rather few and simple identity elements in tRNA(Ser) to determine the amino acid specificity. tRNA(Ser) acceptor stem microhelices can be aminoacylated with serine, which makes this part of the tRNA a valuable tool for investigating the structural motifs in a tRNA(Ser)-seryl-tRNA synthetase complex. A 1.8A-resolution tRNA(Ser) acceptor stem crystal structure was superimposed to a 2.9A-resolution crystal structure of a tRNA(Ser)-seryl-tRNA synthetase complex for a visualization of the binding environment of the tRNA(Ser) microhelix.

Crystal structure of a carbohydrate induced homodimer of phospholipase A2 from Bungarus caeruleus at 2.1A resolution.

This is the first crystal structure of a carbohydrate induced dimer of phospholipase A(2) (PLA(2)). This is an endogenous complex formed between two PLA(2) molecules and two mannoses. It was isolated from Krait venom (Bungarus caeruleus) and crystallized as such. The complete amino acid sequence of PLA(2) was determined using cDNA method. Three-dimensional structure of the complex has been solved with molecular replacement method and refined to a final R-factor of 0.192 for all the data in the resolution range 20.0-2.1A. The presence of mannose molecules in the protein crystals was confirmed using dinitrosalicylic acid test and the molecular weight of the dimer was verified with MALDI-TOF. As indicated by dynamic light scattering and analytical ultracentrifugation the dimer was also stable in solution. The good quality non-protein electron density at the interface of two PLA(2) molecules enabled us to model two mannoses. The mannoses are involved extensively in interactions with protein atoms of both PLA(2) molecules. Some of the critical amino acid residues such as Asp 49 and Tyr 31, which are part of the substrate-binding site, are found facing the interface and interacting with mannoses. The structure of the complex clearly shows that the dimerization is caused by mannoses and it results in the loss of enzymatic activity.

Crystallization of the major cytosolic glutathione S-transferase from Onchocerca volvulus.

Glutathione S-transferases (GSTs) are a family of detoxification enzymes that catalyse the conjugation of glutathione to xenobiotic and endogenous electrophilic compounds, thus facilitating their elimination from cells. The recombinant Onchocerca volvulus GST2 has been expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion technique. Two different crystal forms were grown under identical conditions. They belong to space groups P2(1)2(1)2 and P2(1), respectively. The unit-cell parameters obtained are a = 112.6, b = 84.3, c = 45.1 A for the P2(1)2(1)2 crystal form and a = 51.6, b = 82.3, c = 56.7 A, beta = 95.89 degrees for the P2(1) form. Complete data sets to 2.6 and 1.5 A, respectively, have been collected at 100 K with synchrotron radiation.

Crystallization and preliminary X-ray diffraction studies of the glutathione S-transferase from Plasmodium falciparum.

Glutathione S-transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cells. For high-resolution crystallographic investigations, GST from the human malarial parasite Plasmodium falciparum was overexpressed in bacterial cells and crystallized using hanging-drop vapour diffusion. X-ray intensity data to 2.8 A resolution were collected from an orthorhombic crystal form with unit-cell parameters a = 62.2, b = 88.3, c = 75.3 A. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement.

Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure.

This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).

First look at RNA in L-configuration.

Nucleic acid molecules in the mirror image or L-configuration are unknown in nature and are extraordinarily resistant to biological degradation. The identification of functional L-oligonucleotides called Spiegelmers offers a novel approach for drug discovery based on RNA. The sequence r(CUGGGCGG).r(CCGCCUGG) was chosen as a model system for structural analysis of helices in the L-configuration as the structure of the D-form of this sequence has previously been determined in structural studies of 5S RNA domains, in particular domain E of the Thermus flavus 5S rRNA [Perbandt et al. (2001), Acta Cryst. D57, 219-224]. Unexpectedly, the results of crystallization trials showed little similarity between the D- and the L-forms of the duplex in either the crystallization hits or the diffraction performance. The crystal structure of this L-RNA duplex has been determined at 1.9 A resolution with R(work) and R(free) of 23.8 and 28.6%, respectively. The crystals belong to space group R32, with unit-cell parameters a = 45.7, c = 264.6 A. Although there are two molecules in the asymmetric unit rather than one, the structure of the L-form arranges helical pairs in a head-to-tail fashion to form pseudo-continuous infinite helices in the crystal as in the D-form. On the other hand, the wobble-like G.C(+) base pair seen in the D-RNA analogue does not appear in the L-RNA duplex, which forms a regular double-helical structure with typical Watson-Crick base pairing.

Design of specific peptide inhibitors of phospholipase A2: structure of a complex formed between Russell's viper phospholipase A2 and a designed peptide Leu-Ala-Ile-Tyr-Ser (LAIYS).

Phospholipase A(2) (EC 3.1.1.4) is a key enzyme of the cascade mechanism involved in the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to membrane surfaces and the hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. In order to regulate the production of proinflammatory compounds, a specific peptide inhibitor of PLA(2), Leu-Ala-Ile-Tyr-Ser, has been designed. Phospholipase A(2) from Daboia russelli pulchella (DPLA(2)) and peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) have been co-crystallized. The structure of the complex has been determined and refined to 2.0 A resolution. The structure contains two crystallographically independent molecules of DPLA(2), with one molecule of peptide specifically bound to one of them. The overall conformations of the two molecules are essentially similar except in three regions; namely, the calcium-binding loop including Trp31 (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Of these, the most striking difference pertains to the orientation of Trp31 in the two molecules. The conformation of Trp31 in molecule A was suitable to allow the binding of peptide LAIYS, while that in molecule B prevented the entry of the ligand into the hydrophobic channel. The structure of the complex clearly showed that the OH group of Tyr of the inhibitor formed hydrogen bonds with both His48 N(delta1) and Asp49 O(delta1), while O(gamma)H of Ser was involved in a hydrogen bond with Trp31. Other peptide backbone atoms interact with protein through water molecules, while Leu, Ala and Ile form strong hydrophobic interactions with the residues of the hydrophobic channel.

Crystallisation under microgravity of mistletoe lectin I from Viscum album with adenine monophosphate and the crystal structure at 1.9 A resolution.

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.

Crystal structure of a complex formed between a snake venom phospholipase A(2) and a potent peptide inhibitor Phe-Leu-Ser-Tyr-Lys at 1.8 A resolution.

Phospholipase A(2) is an important enzyme involved in the production of prostaglandins and their related compounds causing inflammatory disorders. Among the several peptides tested, the peptide Phe-Leu-Ser-Tyr-Lys (FLSYK) showed the highest inhibition. The dissociation constant (K(d)) for this peptide was calculated to be 3.57 +/- 0.05 x 10(-9) m. In order to further improve the degree of inhibition of phospholipase A(2), a complex between Russells viper snake venom phospholipase A(2) and a peptide inhibitor FLSYK was crystallized, and its structure was determined by crystallographic methods and refined to an R-factor of 0.205 at 1.8 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) (molecules A and B) and a peptide molecule specifically bound to molecule A only. The two molecules formed an asymmetric dimer. The dimerization caused a modification in the binding site of molecule A. The overall conformations of molecules A and B were found to be generally similar except three regions i.e. the Trp-31-containing loop (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Out of the above three, the most striking difference pertains to the conformation of Trp-31 in the two molecules. The orientation of Trp-31 in molecule A was suitable for the binding of FLSYK, while it disallowed the binding of peptide to molecule B. The structure of the complex clearly shows that the peptide is so placed in the binding site of molecule A that the side chain of its lysine residue interacted extensively with the enzyme and formed several hydrogen bonds in addition to a strong electrostatic interaction with critical Asp-49. The C-terminal carboxylic group of the peptide interacted with the catalytic residue His-48.