Viruses and viral proteins

For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes

Structural and mechanistic investigations on <it>Salmonella typhimurium</it> acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface

<p>Abstract</p> <p>Background</p> <p>Bacteria such as <it>Escherichia coli</it> and <it>Salmonella typhimurium</it> can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism.</p> <p>Results</p> <p>Here we report kinetic characterization of <it>S. typhimurium</it> AckA (<it>St</it>AckA) and structures of its unliganded (Form-I, 2.70 Å resolution) and citrate-bound (Form-II, 1.90 Å resolution) forms. The enzyme showed broad substrate specificity with <it>k</it>_<it>cat</it>/<it>K</it>_<it>m</it> in the order of acetate > propionate > formate. Further, the <it>K</it>_<it>m</it> for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (<it>i.e.</it> ATP synthesis) more efficiently. ATP and Mg²⁺ could be substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn²⁺ and Co²⁺), respectively. Form-I <it>St</it>AckA represents the first structural report of an unliganded AckA. <it>St</it>AckA protomer consists of two domains with characteristic βββαβαβα topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound <it>Methanosarcina thermophila</it> AckA (<it>Mt</it>AckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II <it>St</it>AckA structure showed a drastic change in the conformation of residues 230–300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II <it>St</it>AckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I <it>St</it>AckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes<it>.</it></p> <p>Conclusions</p> <p>The biochemical and structural characterization of <it>St</it>AckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of <it>St</it>AckA led to identification of a putative ligand-binding pocket at the dimeric interface of <it>St</it>AckA with implications for enzymatic function.</p

Dramatic Structural Changes Resulting from the Loss of a Crucial Hydrogen Bond in the Hinge Region Involved in C-Terminal Helix Swapping in SurE: A Survival Protein from Salmonella typhimurium

Domain swapping is an interesting feature of some oligomeric proteins in which each protomer of the oligomer provides an identical surface for exclusive interaction with a segment or domain belonging to another protomer. Here we report results of mutagenesis experiments on the structure of C-terminal helix swapped dimer of a stationary phase survival protein from Salmonella typhimurium (StSurE). Wild type StSurE is a dimer in which a large helical segment at the C-terminus and a tetramerization loop comprising two beta strands are swapped between the protomers. Key residues in StSurE that might promote C-terminal helix swapping were identified by sequence and structural comparisons. Three mutants in which the helix swapping is likely to be avoided were constructed and expressed in E. coli. Three-dimensional X-ray crystal structures of the mutants H234A and D230A/H234A could be determined at 2.1 angstrom and 2.35 angstrom resolutions, respectively. Contrary to expectations, helix swapping was mostly retained in both the mutants. The loss of the crucial D230 OD2- H234 NE2 hydrogen bond (2.89 angstrom in the wild type structure) in the hinge region was compensated by new inter and intra-chain interactions. However, the two fold molecular symmetry was lost and there were large conformational changes throughout the polypeptide. In spite of these changes, the dimeric structure and an approximate tetrameric organization were retained, probably due to the interactions involving the tetramerization loop. Mutants were mostly functionally inactive, highlighting the importance of precise inter-subunit interactions for the symmetry and function of StSurE

Structure of Plasmodium falciparum Triose-phosphate Isomerase-2-Phosphoglycerate Complex at 1.1-Å Resolution

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-Å resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208–211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase- dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue

Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis - distal effects on dimer stability

DatabaseStructural data are available in the Protein Data Bank under the accession numbers <externallink id=''http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3PVF '' type=''url''>3PVF, <externallink id=''http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3PY2 '' type=''url''>3PY2, and <externallink id=''http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3PWA '' type=''url''>3PWA. Structured digital abstract <externallink id=''http://www.uniprot.org/uniprot/Q07412'' type=''url''>Tim <externallink id=''http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407'' type=''url''>binds to <externallink id=''http://www.uniprot.org/uniprot/Q07412'' type=''url''>Tim by <externallink id=''http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114'' type=''url''>x-ray crystallography <externallink id=''http://mint.bio.uniroma2.it/mint/search/interaction.do?interactio nAc=MINT-8149884'' type=''url''>(View interaction)

Symmetry of belladonna mottle virus: rotation function studies

Belladonna mottle virus, a spherical plant virus belonging to the tymovirus group, was crystallized by precipitation with polyethylene glycol 6000 in sodium citrate buffer (pH 5.6). The crystals belong to rhombohedral space group R3 (a \simeq 300 A, \alpha \simeq 60 °) with one molecule in the unit cell, and diffract X-rays to 3.5/~ resolution. Owing to the special interaxial angle of \simeq 60°, the lattice can also be described in terms of a pseudo-face-centred cubic cell. The face-centring vectors of the pseudo cell form the cell edges of the rhombohedral cell. The three-dimensional X-ray diffraction data on these crystals were collected using screenless oscillation photography to a resolution of 6 A. 37 842 independent reflections with {I/ \sigma (I) \geq 2-0} were measured on 51 filn ^pairs. The cell parameters were refined to a = 295.4A and \alpha = 59.86 ° by a postrefinement procedure. A rotation function was calculated using data between 11 and 13 A resolution. The function unambiguously reveals the particle icosahedral symmetry and orientation in the unit cell. The body diagonals of the pseudo-cubic cell are nearly tetrahedral. The icosahedral particle also has a set of tetrahedrally related threefold axes. The particle orientation is such that these two sets can be made coincident by a rotation of \simeq 180 ° about the rhombohedral [111] direction

Crystallization and preliminary X-ray diffraction studies on recombinant diaminopropionate ammonia lyase from Escherichia coli

Diaminopropionate (DAP) ammonia lyase (a PLP-dependent enzyme; EC 4.3.1.15) catalyzes the \alpha,\beta-elimination reaction of both L- and D-\alpha,\beta-diaminopropionate to form pyruvate and ammonia. Escherichia coli DAP ammonia lyase gene was cloned and overexpressed in E. coli and the protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique. Crystals of two different morphologies were obtained, one of which belonged to the tetragonal space group $P4_12_12$ (or $P4_32_12$), with unit-cell parameters a = b = 86.01, c = 209.56 \AA, and the other to the monoclinic space group $P_21$, with unit-cell parameters a = 87.78, b = 94.35, c = 96.02 \AA, = 109.73°. The tetragonal crystals diffracted X-rays to 3.0 \AA resolution, while diffraction from the monoclinic form extended to 2.5 \AA. Complete X-ray diffraction data sets have been collected for both crystal forms

Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

Diaminopropionate ammonialyase (DAPAL), a fold-typeII pyridoxal 5-phosphate-dependent enzyme, catalyzes the ,-elimination of diaminopropionate (DAP) to pyruvate and ammonia. DAPAL was able to utilize both d- and l-DAP as substrates with almost equal efficiency. Mutational analysis of functionally important residues such as Thr385, Asp125 and Asp194 was carried out to understand the mechanism by which the isomers are hydrolyzed. Further, the putative residues involved in the formation of disulfide bond Cys271 and Cys299 were also mutated. T385S, T385D sDAPAL were as active with dl-DAP as substrate as sDAPAL, whereas the later exhibited a threefold increase in catalytic efficiency with d-Ser as substrate. Further analysis of these mutants suggested that DAPAL might follow an anti-E-2 mechanism of catalysis that does not involve the formation of a quinonoid intermediate. Of the two mutants of Asp125, D125E showed complete loss of activity with d-DAP as substrate, whereas the reaction with l-DAP was not affected significantly, demonstrating that Asp125 was essential for abstraction of protons from the d-isomer. By contrast, mutational analysis of Asp194 showed that the residue may not be directly involved in proton abstraction from l-DAP. sDAPAL does not form a disulfide bond in solution, although the position of Cys299 and Cys271 in the modeled structure of sDAPAL favored the formation of a disulfide bond. Further, unlike eDAPAL, sDAPAL could be activated by monovalent cations. Mutation of the cysteine residues showed that Cys271 may be involved in coordinating the monovalent cation, as observed in the case of other fold-typeII enzymes

Stability of belladonna mottle virus particles: the role of polyamines and calcium

The stability of belladonna mottle virus (BDMV) has been studied with respect to elevated pH and to freezing and thawing. BDMV, purified by a modified procedure, was stable at alkaline pH, in contradiction to earlier reports. This difference in the stability could be attributed to the presence of 90 to 140 molecules of spermidine, 20 to 50 molecules of putrescine and 500 to 900 calcium ions in each virus particle. The polyamines could be easily exchanged with other cations such as potassium or caesium and this resulted in a loss of particle stability. These cations may therefore play a role in maintaining the integrity of particle structure. The formation of empty protein shells as a result of freezing and thawing BDMV particles parallels earlier observations on turnip yellow mosaic virus particles

Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue96 in the Plasmodial Enzyme

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residuephenylalanineat this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms