In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors.

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.

Tryptophan as a three-way switch in regulating the function of the secretory signalling glycoprotein (SPS-40) from mammary glands: structure of SPS-40 complexed with 2-methylpentane-2,4-diol at 1.6 A resolution.

The 40 kDa secretory signalling glycoprotein (SPS-40) is the first example with Trp78 in three functional orientations: (i) a resting state with a pinched conformation, (ii) a stacked conformation when bound to hexasaccharide and (iii) an obstructive conformation when inhibited by 2-methylpentane-2,4-diol (MPD). Trp78 is present in the core of the sugar-binding groove. The hexasaccharide N-acetylglucosamine (GlcNAc(6)) has been shown to bind to SPS-40. As a result of this, the conformation of Trp78 alters from the native pinched conformation (chi(1) = -65.5 degrees , chi(2,1) = -78.8 degrees , chi(2,2) = 97.5 degrees ) to the stacked conformation (chi(1) = -170.0 degrees , chi(2,1) = -114.3 degrees , chi(2,2) = 61.6 degrees ). Further binding experiments showed that saccharide binding does not occur in the presence of 20% MPD. The crystal structure determination of the complex of SPS-40 with MPD revealed the presence of two MPD molecules in the sugar-binding groove. The very tightly bound MPD molecules at subsites -2 and -1 induced an unexpected and a rarely observed conformation of Trp78 (chi(1) = 55.9 degrees , chi(2,1) = 90.2 degrees , chi(2,2) = -88.9 degrees ) which is termed an obstructive conformation. The binding of MPD molecules also twisted the side chains of Glu269 and Ile272 considerably. These residues are also part of the sugar-binding groove. The observed obstructive conformation of the side chain of Trp78 in the present structure is the exact opposite of the stacked conformation. This rarely observed conformation is stabilized by a number of hydrogen bonds between Trp78 and Asn79 through water molecules W49, W229, W269, W547 and W557.

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.

De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography.

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 1.9 Šresolution using automatic model-building tools.

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.

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.

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop.

The synthetic RNA fragment 5'-CUGGGCGG(GCGA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79-97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 A and refined to an R-value of 24.1% also represents the first X-ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments.

Crystal structure of vipoxin at 2.0 A: an example of regulation of a toxic function generated by molecular evolution.

Vipoxin is the main toxic component in the venom of the Bulgarian snake Vipera ammodytes meridionalis, the most toxic snake in Europe. Vipoxin is a complex between a toxic phospholipase A2 (PLA2) and a non-toxic protein inhibitor. The structure is of genetic interest due to the high degree of sequence homology (62%) between the two functionally different components. The structure shows that the formation of the complex in vipoxin is significantly different to that seen in many known structures of phospholipases and contradicts the assumptions made in earlier studies. The modulation of PLA2 activity is of great pharmacological interest, and the present structure will be a model for structure-based drug design.

[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.

[A comparative structure-function analysis and molecular mechanism of action of endonucleases from Serratia marcescens and Physarum polycephalum]. / Sravnitel'nyi strukturno-funktsional'nyi analiz éndonukleaz Serratia marcescens i Physarum polycephalum i molekuliarnyi mekhanizm ikh deistviia.

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.

Crystal structure of the peptidoglycan recognition protein at 1.8 A resolution reveals dual strategy to combat infection through two independent functional homodimers.

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.

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.

Purine activity of RNase T1RV is further improved by substitution of Trp59 by tyrosine.

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.

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.

Structure of an RNA duplex with an unusual G.C pair in wobble-like conformation at 1.6 A resolution.

The structure of the RNA duplex r(CUGGGCGG).r(CCGCCUGG) has been determined at 1.6 A resolution and refined to a final R factor of 18.3% (R(free) = 24.1%). The sequence of the RNA fragment resembles domain E of Thermus flavus 5S rRNA. A previously undescribed wobble-like G.C base-pair formation is found. Owing to the observed hydrogen-bond network, it is proposed that the cytosine is protonated at position N3. The unusual base-pair formation is presumably strained by intermolecular interactions. In this context, crystal packing and particular intermolecular contacts may have direct influence on the three-dimensional structure. Furthermore, this structure includes two G.U wobble base pairs in tandem conformation, with the purines forming a so-called 'cross-strand G stack'.

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.

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.