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The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine. [ABSTRACT FROM AUTHOR]

In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands. The authors present an investigation of Tm variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES [pH 7.5], 150 mM NaCl) permitted estimation of Tm for 58 of 61 Plasmodium proteins tested. However, with several proteins, Tm could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. The authors conclude that buffer optimization to minimize variability in Tm measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal. [ABSTRACT FROM AUTHOR]

The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed α-helical region at one side of the canonical phospholipid-binding site. Re-examination of previously determined PEBP structures suggests that the P. vivax protein and yeast carboxypeptidase Y inhibitor may represent a structurally distinct subfamily of the diverse PEBP-sequence family. [ABSTRACT FROM AUTHOR]

A complex molecular motor empowers substrate-dependent motility and host cell invasion in malaria parasites. The interaction between aldolase and the transmembrane adhesin thrombospondin-related anonymous protein (TRAP) transduces the motor force across the parasite surface. Here, we analyzed this interaction by using state-of-the-art flexible docking. Besides algorithms to account for induced fit in the side-chains of the Plasmodium falciparum aldolase (PfAldo) structure, we used additional in silico receptors modeled upon crystallographic structures of evolutionarily related aldolases to incorporate enzyme backbone flexibility, and to overcome structure inaccuracies due to the relatively low resolution (3.0 Å) of the genuine PfAldo structure. Our results indicate that, in spite of multiple intermolecular contacts, only the six C-terminal residues of the TRAP cytoplasmic tail bind in an ordered manner to PfAldo. This portion of TRAP targets the PfAldo active site, with its n-1 Trp residue, which is essential for this interaction, buried within the PfAldo catalytic pocket. Docking of a TRAP peptide bearing a Trp to Ala mutation rendered the lower energy configurations either bound weakly outside the active site or not bound to PfAldo at all. The position of the bound TRAP peptide, and particularly the close proximity between the carbonyl of its n-2 Asp residue and the experimentally determined position of the phosphate-6 group of fructose 1,6-phosphate bound to mammalian aldolases, predicts an inhibitory effect of TRAP on catalysis. Enzymatic and TRAP-binding assays using mutant PfAldo molecules strongly support the overall structural model. These results might provide the initial framework for the identification of novel antiparasitic compounds. Proteins 2007. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

EpsE is a cytoplasmic component of the type II secretion system in Vibrio cholerae. Through ATP hydrolysis and an interaction with the cytoplasmic membrane protein EpsL, EpsE supports secretion of cholera toxin across the outer membrane. In this study, we have determined the effect of the cytoplasmic domain of EpsL (cyto-EpsL) and purified phospholipids on the ATPase activity of EpsE. Acidic phospholipids, specifically cardiolipin, bound the copurified EpsE/cyto-EpsL complex and stimulated its ATPase activity 30–130-fold, whereas the activity of EpsE alone was unaffected. Removal of the last 11 residues (residues 243–253) from cyto-EpsL prevented cardiolipin binding as well as stimulation of the ATPase activity of EpsE. Further mutagenesis of the C-terminal region of the EpsL cytoplasmic domain adjacent to the predicted transmembrane helix suggested that this region participates in fine tuning the interaction of EpsE with the cytoplasmic membrane and influences the oligomerization state of EpsE thereby stimulating its ATPase activity and promoting extracellular secretion in V. cholerae. [ABSTRACT FROM AUTHOR]

The structure of ribose 5-phosphate isomerase from Plasmodium falciparum, PFE0730c, has been determined by molecular replacement at 2.09 Å resolution. The enzyme, which catalyzes the isomerization reaction that interconverts ribose 5-phosphate and ribulose 5-phosphate, is a member of the pentose phosphate pathway. The P. falciparum enzyme belongs to the ribose 5-­phosphate isomerase A family, Pfam family PF06562 (DUF1124), and is structurally similar to other members of the family. [ABSTRACT FROM AUTHOR]

The structure of a conserved hypothetical protein, PlasmoDB sequence MAL13P1.257 from Plasmodium falciparum, Pfam sequence family PF05907, has been determined as part of the structural genomics effort of the Structural Genomics of Pathogenic Protozoa consortium. The structure was determined by multiple-wavelength anomalous dispersion at 2.17 Å resolution. The structure is almost entirely β-sheet; it consists of 15 β-strands and one short 310-helix and represents a new protein fold. The packing of the two monomers in the asymmetric unit indicates that the biological unit may be a dimer. [ABSTRACT FROM AUTHOR]

The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD) using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 ( Rfree = 0.229) at 1.60 Å resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria use the type II secretion system to transport a large number of secreted proteins from the periplasmic space into the extracellular environment. Many of the secreted proteins are major virulence factors in plants and animals. The components of the type II secretion system are located in both the inner and outer membranes where they assemble into a multi-protein, cell-envelope spanning, complex. This review discusses recent progress, particularly newly published structures obtained by X-ray crystallography and electron microscopy that have increased our understanding of how the type II secretion apparatus functions and the role that individual proteins play in this complex system. [ABSTRACT FROM AUTHOR]

The crystal structure of Pfal009167AAA, a putative ribulose 5-phosphate 3-epimerase (PfalRPE) from Plasmodium falciparum, has been determined to 2 Å resolution. RPE represents an exciting potential drug target for developing antimalarials because it is involved in the shikimate and the pentose phosphate pathways. The structure is a classic TIM-barrel fold. A coordinated Zn ion and a bound sulfate ion in the active site of the enzyme allow for a greater understanding of the mechanism of action of this enzyme. This structure is solved in the framework of the Structural Genomics of Pathogenic Protozoa (SGPP) consortium. Proteins 2006. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Purine metabolism in the parasite Plasmodium has been identified as a promising target for antimalarial therapies. Purine nucleoside phosphorylase (PNP) is part of a salvage pathway for the biosynthesis of purines, which are essential for parasite survival. Two crystal structures of PNP from Plasmodium falciparum (PƒPNP) in two space groups, each with a single subunit in the asymmetric unit, are described here. One structure, refined to 2.4 Å, has an empty nucleoside-binding site and a sulfate ion bound in the phosphate-binding pocket. The second structure, refined to 2.0 Å, has the substrate inosine bound to the active centre. Structure comparison reveals alterations in the active site upon ligand binding. The new structures presented here specifically highlight the likely roles of Asp206 and two loops flanking the active site: the β7-α6 loop (residues ∼161–169) and the β9-α8 loop (residues ∼208–223). Comparison with PNP in complex with transition-state inhibitors suggests that the purine substrate moves towards the phosphate substrate, rather than vice versa, upon forming the transition state. The single-substrate-containing PƒPNP structures also appear to be more flexible than PƒPNP bound to inhibitors. Together, these structures serve as a basis for better understanding of ligand binding and mechanism that can be further exploited to optimize the specificity of anti-PƒPNP drugs. [ABSTRACT FROM AUTHOR]

Precise control of substrate binding and release is essential for molecular chaperones to exert their protective function in times of stress. The mechanisms used are diverse and have been difficult to unravel. Escherichia coli heat-shock protein 31 (Hsp31) is a recent addition to the known complement of eubacterial chaperones. Crystallographic studies have revealed the presence of a hydrophobic bowl at the Hsp31 dimer interface and shown that the linker region connecting the two structural domains within each subunit is disordered. Together with a neighboring flexible loop, the linker caps a hydrophobic area adjacent to the bowl. Using a collection of Hsp31 mutants, we show that although both bowl and linker-loop-shielded residues participate in substrate binding, the latter are critical for protein capture at high temperature. Linker immobilization via an artificial disulfide bridge abolishes chaperone activity at elevated temperatures by precluding exposure of the underlying hydrophobic domain. We conclude that Hsp31 uses its linker-loop region as a thermally activated gate to control nonnative protein annealing to a high-affinity substrate-binding site. This simple yet efficient strategy to capture partially folded proteins under heat-shock conditions may be shared by other folding modulators. [ABSTRACT FROM AUTHOR]

In the quest to develop drugs against traveller's diarrhoea and cholera, the structure of the B pentamer of heat-labile enterotoxin (LT) complexed with a new receptor-binding antagonist, m-carboxyphenyl-α-D-galactopyranoside, has been determined. The high resolution obtained for this structure allowed anisotropic refinement of the model. it was also now possible to confirm at a near-atomic resolution the structural similarity between the B subunits of LT and the closely related cholera toxin (CT), including the similarity in deviations of planarity of the same peptide unit in LT and CT. The structure of the LT complex clearly revealed different conformations for the m-carboxyphenyl moiety of the ligand in the five B subunits of LT, while the binding modes of the well defined galactopyranoside moieties were identical. in two binding sites the m-carboxyphenyl moiety displayed no significant electron density, demonstrating significant flexibility of this moiety. in a third binding site the m-carboxyphenyl moiety could be modelled unambiguously into the density. The two remaining binding sites were involved in crystal packing contacts and the density for the ligands in these two binding sites clearly revealed different binding modes, of which one conformation was identical to and one completely different from the conformation of m-carboxyphenyl-galactopyranoside in the third subunit. The multiple binding modes observed in the crystal may represent the ensemble of conformations of m-carboxyphenyl-α-D-galactopyranoside complexed to LT in solution. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 Å resolution by X-ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55-residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two β-strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N-terminal domain being wedge-shaped and the C-terminal domain flat. Docking studies suggest that differences in domain shape enable the N-terminal, but not C-terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N-terminal domain of antistasin, comprising residues 15–17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C-terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain. [ABSTRACT FROM AUTHOR]

Partial sequences of the dihydrolipoyl transacetytase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over-expressed in E. coli TG2. A high expression of a homogeneous protein was only detectable for E2p mutants consisting of the catalytic domain and the alanine-proline-rich sequence between a putative binding region for the peripheral components and the catalytic domain(apa-4). Most of the catalytic domain from A. vinelandii without the apa-4 sequence was degraded intracellularly, probably due to incorrect folding. Fusion proteins of six amino acids from β-galactosidase, the apa-4 region and the catalytic domains of ,4, vinelandii or E. coli E2p could be highly purified. Both catalytic domains were assembled in 24-subtmit structures with a molecular mass of approximately 670 kDa. The expression of catalytic domain from A. vinelandii E2p is more than twice as high as found for wild-type E2p. This can be explained by intracellular degradation of overexpressed wild-type E2p, whereas the catalytic domains are stable against proteolysis/n vivo and in vitro. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and dihydrolipoamide dehydrogenase (E3) with the catalytic domains was studied, using gel filtration on Superose-6 and sedimentation velocity experiments. No binding of either E1p or E3 to the catalytic domain of either organism was detectable. Crystals of the catalytic domain of A. vinelandii E2p could ix: grown to a maximum size of 0.6 × 0.6 times; 0.4 mm. They diffract up to a resolution of 0.28 nm. [ABSTRACT FROM AUTHOR]

The structure of the enzyme p-hydroxybenzoate hydroxylase was determined to a resolution of 0.25 nm [Wierenga et al. (1979) J. Mol. Biol. 131, 53-73] with crystals belonging to space group C2221. Subsequently it was impossible to repeat the growth of this crystal form and only poor quality tetragonal crystals could be obtained. We have thoroughly investigated this problem and found that Cibacron-blue-purified enzyme appears to be heterogeneous with respect to aggregation state and Cys-116 oxidation. Most importantly, it could be firmly established that C2221, crystals can only be grown from purely dimeric p-hydroxybenzoate hydroxylase possessing an intact SH group. Ion-exchange chromatography on DEAE-Sepharose can successfully remove those forms of the enzyme which impede successful crystallizatioun. Sulfite and dithiothreitol improve crystallization by dissociating the enzyme oligomers into dimers; sulfite especially gives excellent results. [ABSTRACT FROM AUTHOR]

Cholera toxin (CT) produced by Vibrio cholerae and heat-labile enterotoxin (LT-I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP-ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti-diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT-I are known to be relatively temperature sensitive. To improve the thermostability of LT-I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT-I has been determined to 2.0 Å resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT-I by 6°C. The enhancement in thermostability could make this disulfide bond variant of LT-I of considerable interest for the design of enterotoxin-based vaccines. [ABSTRACT FROM AUTHOR]

Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 Å resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the first domain (positions 1·175) of Panulirus interruptus hemocyanin subunit a has been determined. The sequence of residues 1·158 (18-kDa fragment obtained by limited proteolysis) was derived from peptides obtained by digestion of this fragment with CNBr and trypsin and by subdigestion of these peptides with other enzymes. The peptides were sequenced automatically or manually. The amino acid sequence has been fitted into the electron-density map at 0.32-nm resolution. The residues of domain 1 are folded into a larger, mainly helical, globular part, containing one disulfide bridge, and a smaller part near the molecular twofold axis. The latter part consists of an α helix and a β strand which contains a covalently attached carbohydrate moiety. The sites susceptible to limited proteolytic cleavage of the subunit are discussed. Comparison of the N-terminal sequence with those of other arthropod hemocyanins revealed, besides an N- terminal extension of five residues, the presence of a 21-residue loop (positions 22–42) in the crustacean sequences. This loop contains helix 1.2, a less defined region in the electron-density map. It is absent in chelicerate sequences. Strong evidence is presented that: (a) the structure of the first 21 residues (including helix 1.1) is the same in all arthropod hemocyanins with known amino acid sequence; (b) a stretch containing about 15 residues (including part of helix 1.3) following the 21-residue loop has a different structure in crustaceans and chelicerates; (c) the rest of domain 1 has the same structure again. It is shown that all conserved residues are in the contact region with the other two domains. [ABSTRACT FROM AUTHOR]

The enzyme methylamine dehydrogenase or primary-amine :(acceptor) oxidoreductase (deaminating) (EC 1.4.99,3) was purified from the bacterium Thiobacillus versutus to homogeneity, as judged by polyacrylamide gel electrophoresis. The native enzyme has a Mr of 123 500 and contains four subunits arranged in a &alpha2β2 configuration, the light and heavy subunits having a Mr of 12900 and 47500 respectively. The isoelectric point is 3.9. The purified enzyme was crystallized from 37 -- 42% saturated ammonium sulphate in 0.1 M sodium acetate buffer, pH 5.0. The space group is P3121 or P3221, with one α2β2 molecule in the asymmetric unit. The cell dimensions are: a = b = 13.01 nm; c = 10.40 nm. The X-ray diffraction pattern extends to at least 0.25-nm resolution. [ABSTRACT FROM AUTHOR]

The binding of sulfate, selenate and thiosulfate by the sulfur-transferase rhodanese (EC 2.8.1.1) in the crystalline state has been studied by X-ray analysis at resolutions between 0.23 nm and 0.4 nm. The three ions appear to occupy a common site between the Nη atoms of Arg-29 and the main-chain NH group of Glu-148 at the surface of the enzyme molecule. A second binding site for the three ions is situated at the entrance to the active centre, between the side chains of Arg-186 and Lys-249. Selenate and thiosulfate are bound equally well at both anion-binding sites. Sulfate, however, binds better at the first position, near Arg-29, than at the second site near Arg-186. In the complex of sulfur-rhodanese with thiosulfate, the outer sulfur atom of the anion near the active centre points towards the extra sulfur atom which is bound as a persulfide to the Sγ of the essential Cys.247. The distance between the outer sulfur atom of the thiosulfate ion and the persulfide sulfur atom appears to be about 0.3nm. The thiosulfate difference Fourier also shows a distinct, localized con formational change involving residues 71, 72 and 249. This is the result of the replacement of an ammonium ion in the sulfate and selenate media by a sodium ion in the sodium thiosulfate solution. Rhodanese is apparently able to accomodate ions with different radii at this cation-binding site by minor structural alterations. [ABSTRACT FROM AUTHOR]