Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and monitoring antiviral resistant strains.

Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types.

Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens.

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

Structure of the fiber head of Ad3, a non-CAR-binding serotype of adenovirus.

Adenoviruses of serotype Ad3 (subgenus B) use a still-unknown host cell receptor for viral attachment, whereas viruses from all other known subgenera use the coxsackie and adenovirus receptor (CAR). The receptor binding domain (head) of the Ad3 fiber protein has been expressed in Escherichia coli inclusion bodies. After denaturation and renaturation using a rapid dilution method, crystals of trimeric head were obtained. The 1.6 A resolution X-ray structure shows a strict conservation of the beta-sheet scaffold of the protein very similar to the head structures of the CAR-binding serotypes Ad2, Ad5, and Ad12. The conformation of the loops is different, with the exception of the AB loop, which forms the center of the interface in the Ad12-CAR complex structure. The structure explains why a mutation in Ad5 of one residue in the AB loop to glutamic acid, as in Ad3, abrogates binding to CAR. It is possible that the Ad3 receptor binding site is nevertheless situated similar to the CAR binding site, although it cannot be excluded that other regions of the relatively hydrophobic head surface may be used.

Crystal structure of a monocotyledon (maize ZMGlu1) beta-glucosidase and a model of its complex with p-nitrophenyl beta-D-thioglucoside.

The maize beta-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Angstrom resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl beta-D-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.

Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer.

The amino-terminal domain of influenza A virus matrix protein (residues 1-164) was crystallized at pH 7 into a new crystal form in space group P1. This packing of the protein implies that M1(1-164) was monomeric in solution when it crystallized. Otherwise, the structure of the M1 fragment in the pH 7 crystals was the same as the monomers in crystals formed at pH 4 where crystal packing resulted in dimer formation [B. Sha and M. Luo, 1997, Nature Struct. Biol. 4, 239-244]. Analysis of intact M1 protein, the N-terminal domain, and the remaining C-terminal fragment (residues 165-252) in solution also showed that the N-terminal domain was monomeric with the same dimensions as determined from the crystal structure. Intact M1 protein was also monomeric but with an elongated shape due to the presence of the C-terminal part. Circular dichroism showed that the C-terminal part of M1 contained helical structure. A model for soluble M1 is presented, based on the assumption that the C-terminal domain is spherical, in which the N- and C-terminal domains are connected by a linker sequence which is available for proteolytic attack.

High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base.

Myrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio-beta-d-glucosides (glucosinolates) considered part of the plant defense system. Although O-glycosidases are ubiquitous, myrosinase is the only known S-glycosidase. Its active site is very similar to that of retaining O-glycosidases, but one of the catalytic residues in O-glycosidases, a carboxylate residue functioning as the general base, is replaced by a glutamine residue. Myrosinase is strongly activated by ascorbic acid. Several binary and ternary complexes of myrosinase with different transition state analogues and ascorbic acid have been analyzed at high resolution by x-ray crystallography along with a 2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors, d-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a mimic of the enzyme-substrate complex. Ascorbate binds to a site distinct from the glucose binding site but overlapping with the aglycon binding site, suggesting that activation occurs at the second step of catalysis, i.e. hydrolysis of the glycosyl enzyme. A water molecule is placed perfectly for activation by ascorbate and for nucleophilic attack on the covalently trapped 2-fluoro-glucosyl-moiety. Activation of the hydrolysis of the glucosyl enzyme intermediate is further evidenced by the observation that ascorbate enhances the rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion that ascorbic acid substitutes for the catalytic base in myrosinase.

Tetrameric coiled coil domain of Sendai virus phosphoprotein.

The high resolution X-ray structure of the Sendai virus oligomerization domain reveals a homotetrameric coiled coil structure with many details that are different from classic coiled coils with canonical hydrophobic heptad repeats. Alternatives to the classic knobs-into-holes packing lead to differences in supercoil pitch and diameter that allow water molecules inside the core. This open and more hydrophilic structure does not seem to be destabilized by mutations that would be expected to disrupt classic coiled coils.

Structural changes in a cryo-cooled protein crystal owing to radiation damage.

The high intensity of third-generation X-ray sources, along with the development of cryo-cooling of protein crystals at temperatures around 100 K, have made it possible to extend the diffraction limit of crystals and to reduce their size. However, even with cryo-cooled crystals, radiation damage becomes a limiting factor. So far, the radiation damage has manifested itself in the form of a loss of overall diffracted intensity and an increase in the temperature factor. The structure of a protein (myrosinase) after exposure to different doses of X-rays in the region of 20 x 10(15) photons mm(-2) has been studied. The changes in the structure owing to radiation damage were analysed using Fourier difference maps and occupancy refinement for the first time. Damage was obvious in the form of breakage of disulfide bonds, decarboxylation of aspartate and glutamate residues, a loss of hydroxyl groups from tyrosine and of the methylthio group of methionine. The susceptibility to radiation damage of individual groups of the same kind varies within the protein. The quality of the model resulting from structure determination might be compromised owing to the presence of radiolysis in the crystal after an excessive radiation dose. Radiation-induced structural changes may interfere with the interpretation of ligand-binding studies or MAD data. The experiments reported here suggest that there is an intrinsic limit to the amount of data which can be extracted from a sample of a given size.

On the domain structure and the polymerization state of the sendai virus P protein.

The phosphoproteins (P) of paramyxoviruses and rhabdoviruses are cofactors of the viral polymerase (L) and chaperones of soluble nucleoprotein preventing its polymerization and nonspecific binding to cellular RNA. The primary sequences of six paramyxovirus P proteins were compared, and although there was virtually no sequence similarity, there were two regions with similar secondary structure predictions in the C-terminal part of P: the predicted multimerization domain and the X-protein, the sequence that binds to N in the N:RNA template. The C-terminal part of the Sendai virus P protein, the multimerization domain including the binding site for the polymerase, and the X-protein were expressed in Escherichia coli. All three polypeptides folded with secondary structures similar to those predicted. The C-terminal part of P is a very elongated molecule with most of its length encompassing the multimerization domain. Both the multimerization domain and the C-terminal part of P were found to form tetramers, whereas the X-protein was monomeric.

The crystal structure of the minus-end-directed microtubule motor protein ncd reveals variable dimer conformations.

BACKGROUND: The kinesin superfamily of microtubule-associated motor proteins are important for intracellular transport and for cell division in eukaryotes. Conventional kinesins have the motor domain at the N terminus of the heavy chain and move towards the plus end of microtubules. The ncd protein is necessary for chromosome segregation in meiosis. It belongs to a subfamily of kinesins that have the motor domain at the C terminus and move towards the minus end of microtubules. RESULTS: The crystal structure of dimeric ncd has been obtained at 2.9 A resolution from crystals with the C222(1) space group, with two independent dimers per asymmetric unit. The motor domains in these dimers are not related by crystallographic symmetry and the two ncd dimers have significantly different conformations. An alpha-helical coiled coil connects, and interacts with, the motor domains. CONCLUSIONS: The ncd protein has a very compact structure, largely due to extended interactions of the coiled coil with the head domains. Despite this, we find that the overall conformation of the ncd dimer can be rotated by as much as 10 degrees away from that of the twofold-symmetric archetypal ncd. The crystal structures of conventional kinesin and of ncd suggest a structural rationale for the reversal of the direction of movement in chimeric kinesins.

ID14 'Quadriga', a Beamline for Protein Crystallography at the ESRF.

The ESRF undulator beamline ID14 'Quadriga' is dedicated to monochromatic macromolecular crystallography. Using two undulators with 23 mm and 42 mm periods and a minimum gap of 16 mm installed on a high-beta section, it will provide high-brilliance X-ray beams at around 13.5 keV, as well as a wide tuneability between 6.8 and 40 keV. Based on the Troika concept, this beamline has four simultaneously operating experimental stations: three side stations, EH1, EH2 and EH3, using thin diamond crystals, and an end station, EH4, with a fast-scan double-crystal monochromator. Station EH3 has a kappa-diffractometer, and an off-line Weissenberg camera with a large 80 x 80 cm active area combined with a 2048 x 2048 CCD detector. During data collection the image plates are placed and removed by a robot located inside the hutch using a cassette system. After data collection the image plates are scanned with an off-line drum scanner. Station EH4 is designed for MAD applications, including Xe K-edge anomalous experiments, and is equipped with a 2048 x 2048 CCD detector on a pseudo 2theta arm. A common graphical user interface and a database will be available to cover all aspects of data collection, including strategy optimization. First results on the performance of the optics elements and initial crystallographic results are presented.

The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase.

BACKGROUND: Myrosinase is the enzyme responsible for the hydrolysis of a variety of plant anionic 1-thio-beta-D-glucosides called glucosinolates. Myrosinase and glucosinolates, which are stored in different tissues of the plant, are mixed during mastication generating toxic by-products that are believed to play a role in the plant defence system. Whilst O-glycosidases are extremely widespread in nature, myrosinase is the only known S-glycosidase. This intriguing enzyme, which shows sequence similarities with O-glycosidases, offers the opportunity to analyze the similarities and differences between enzymes hydrolyzing S- and O-glycosidic bonds. RESULTS: The structures of native myrosinase from white mustard seed (Sinapis alba) and of a stable glycosyl-enzyme intermediate have been solved at 1.6 A resolution. The protein folds into a (beta/alpha)8-barrel structure, very similar to that of the cyanogenic beta-glucosidase from white clover. The enzyme forms a dimer stabilized by a Zn2+ ion and is heavily glycosylated. At one glycosylation site the complete structure of a plant-specific heptasaccharide is observed. The myrosinase structure reveals a hydrophobic pocket, ideally situated for the binding of the hydrophobic sidechain of glucosinolates, and two arginine residues positioned for interaction with the sulphate group of the substrate. With the exception of the replacement of the general acid/base glutamate by a glutamine residue, the catalytic machinery of myrosinase is identical to that of the cyanogenic beta-glucosidase. The structure of the glycosyl-enzyme intermediate shows that the sugar ring is bound via an alpha-glycosidic linkage to Glu409, the catalytic nucleophile of myrosinase. CONCLUSIONS: The structure of myrosinase shows features which illustrate the adaptation of the plant enzyme to the dehydrated environment of the seed. The catalytic mechanism of myrosinase is explained by the excellent leaving group properties of the substrate aglycons, which do not require the assistance of an enzymatic acid catalyst. The replacement of the general acid/base glutamate of O-glycosidases by a glutamine residue in myrosinase suggests that for hydrolysis of the glycosyl-enzyme, the role of this residue is to ensure a precise positioning of a water molecule rather than to provide general base assistance.

Structures of two classes of MHC molecules elucidated: crucial differences and similarities.

New structures of MHC molecules have significantly improved our understanding of molecular recognition in cellular immunology. Highlights include the first structure of a class II MHC molecule, complexed with a viral peptide and with a bacterial superantigen. A structure of an MHC-like Fc receptor is expected soon. Interesting comparisons can now be made between the recognition properties of MHC and MHC-like proteins.

Crystal structure at 2.2 A resolution of the MHC-related neonatal Fc receptor.

The three-dimensional structure of the rat neonatal Fc receptor (FcRn) is similar to the structure of molecules of the major histocompatibility complex (MHC). The counterpart of the MHC peptide-binding site is closed in FcRn, making the FcRn groove incapable of binding peptides. A dimer of FcRn heterodimers seen in the crystals may represent a receptor dimer that forms when the Fc portion of a single immunoglobulin binds. An alternative use of the MHC fold for immune recognition is indicated by the FcRn and FcRn/Fc co-crystal structures.

Crystal structure of the complex of rat neonatal Fc receptor with Fc.

The neonatal Fc receptor (FcRn) transports maternal immunoglobulin G (IgG) to the bloodstream of the newborn. FcRn is structurally similar to class I major histocompatibility complex (MHC) molecules, despite differences in the ligands they bind (the Fc portion of IgG and antigenic peptides, respectively). A low-resolution crystal structure of the complex between FcRn and Fc localizes the binding site for Fc to the side of FcRn, distinct from the tops of the alpha 1 and alpha 2 domains which serve as the peptide and T-cell receptor binding sites in class I molecules. FcRn binds to Fc at the interface between the Fc CH2 and CH3 domains, which contains several histidine residues that could account for the sharply pH-dependent FcRn/IgG interaction. A dimer of FcRn heterodimers observed in the co-crystals and in the crystals of FcRn alone could be involved in binding Fc, correlating with the 2:1 binding stoichiometry between FcRn and IgG (ref. 4) and suggesting an unusual orientation of FcRn on the membrane.

Calcium is needed for the thermostability of influenza B virus neuraminidase.

The activity and stability of influenza virus neuraminidase is known to depend on the presence of calcium ions. The atomic structure of the tetrameric neuraminidase head shows two distinct Ca2+ binding sites, one with low affinity on the molecular fourfold symmetry axis and one with high affinity close to the active site in each of the monomers. Here we show that Ca is essential for the thermostability of the isolated neuraminidase tetramer. Inactivation of Ca-free neuraminidase at high temperatures is accompanied by changes in protein structure leading to protease sensitivity. More than one Ca ion per tetramer is involved in stabilization, suggesting a role for the high affinity Ca binding site and the cooperative stabilization of the subunits. Sites which are located close to the fourfold axis of the neuraminidase tetramer and which are able to bind a variety of different metal ions are also described.

Influenza B virus neuraminidase can synthesize its own inhibitor.

BACKGROUND: Neuraminidase, one of the two surface glycoproteins of influenza virus, cleaves terminal sialic acid residues from glycolipids or glycoproteins. Its crystal structure is known at high resolution, but the mechanism of glycosyl hydrolysis remains unclear. RESULTS: We have determined the crystal structure at 1.8 A resolution of two complexes of influenza B/Beijing neuraminidase containing either the reaction product, sialic acid, or the transition state analogue inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). The sialic acid is bound in a distorted 'boat' conformation closely resembling that of bound DANA, stabilized by a conserved tyrosine residue (Tyr408). This distortion also gives rise to a suicidal side reaction that converts sialic acid to DANA at a low rate. CONCLUSIONS: The mechanism of neuraminidase action is distinct from that of other known glycosyl hydrolases. Substrate distortion appears to be the driving force in glycosyl bond hydrolysis and the proton required for catalysis can probably be donated by water, rather than by residues in the active site, thus allowing the enzyme to operate at high pH. The side reaction converting sialic acid to DANA appears reasonably favourable, and it is unclear how this is minimized by the enzyme.

Comparison of structure and sequence of influenza B/Yamagata and B/Beijing neuraminidases shows a conserved "head" but much greater variability in the "stalk" and NB protein.

In 1988 the B/Yamagata/16/88 influenza virus appeared, which was antigenically quite different from the other B viruses circulating at that time. Neuraminidase heads of this strain were purified, crystallized, and analyzed by X-ray crystallography and compared with the structure of B/Beijing/1/87 neuraminidase. Only two point mutations could be detected in the "head" of the molecule leading to local rearrangement of amino acid side-chains. The nucleotide sequence, however, revealed significant differences in the stalk region and in the NB protein.

The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid.

Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R-factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co-ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.