Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes.

Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labeled cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cell responses is proposed to be the protein loading with a sterol molecule.

Crystal structure of a fungal elicitor secreted by Phytophthora cryptogea, a member of a novel class of plant necrotic proteins.

BACKGROUND: Elicitins form a novel class of plant necrotic proteins which are secreted by Phytophthora and Pythium fungi, parasites of many economically important crops. These proteins induce leaf necrosis in infected plants and elicit an incompatible hypersensitive-like reaction, leading to the development of a systemic acquired resistance against a range of fungal and bacterial plant pathogens. No crystal structures of this class of protein are available. The crystal structure determination of beta-cryptogein (CRY), secreted by Phytophthora cryptogea, was undertaken to identify structural features important for the necrotic activity of elicitins. RESULTS: The structure of CRY was determined using the multiwavelength anomalous diffraction technique and refined to 2.2 A resolution. The overall structure has a novel fold consisting of six alpha helices and a beak-like motif, whose sequence is highly conserved within the family, composed of an antiparallel two-stranded beta sheet and an omega loop. This motif is assumed to be a major recognition site for a putative receptor and/or ligand. Two other distinct binding sites seem to be correlated to the level of necrotic activity of elicitins. CONCLUSIONS: The determination of the crystal structure of a member of the elicitin family may make it possible to separate the activity that causes leaf necrosis from that inducing systemic acquired resistance to pathogens, making it feasible to engineer a non-toxic elicitin that only elicits plant defences. Such studies should aid the development of non-toxic agricultural pest control.

Crystallographic study at 2.5 A resolution of the interaction of methionyl-tRNA synthetase from Escherichia coli with ATP.

The crystal structure of the tryptic fragment of the methionyl-tRNA synthetase from Escherichia coli, complexed with ATP, has been refined to a crystallographic R-factor of 0.220, at 2.5 A resolution (for 4433 protein atoms). In the last stages of the refinement, the simulated annealing refinement method was fully applied, contributing to a drastic improvement of the model and the identification of the missing atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.021 A and for angle distances is 0.054 A. The position of the zinc ion has been confirmed and is located near the active site. The tryptic fragment is composed of two globular domains. The first domain, from the N terminus to Thr360, contains a nucleotide-binding fold into which two long polypeptides of 101 and 70 residues are inserted. The nucleotide-binding fold is strengthened by the presence of the zinc ion in the vicinity of the active site. The second domain, up to Pro526, is mainly alpha-helical. The C-terminal polypeptide, Phe527 to Lys551, folds back towards the first domain, making a link between the two domains. The heptapeptide 528-534 partly shapes a deep cavity that plunges into the central core of the nucleotide-binding fold, where the ATP molecule is located. The adenine ring, deeply buried in the bottom of the cleft, is blocked between the first helix HA, and the strands A and D of the beta-sheet and makes no polar interaction with the enzyme. The 2' and 3' hydroxyl groups of the ribose, whose conformation is C2' endo, interact with the main-chain carbonyl oxygen atoms of Ile231 and Glu241, respectively. The side-chain nitrogen atom of Lys142 is at hydrogen-bonding distance from the ring oxygen O-4' of the ribose. One of the alpha-phosphate oxygen atoms and one of the gamma-phosphate oxygen atoms interact with the imidazole ring of His21, which is well conserved in many of the known synthetases; this indicates a possible crucial role for this residue in binding ATP. The beta-phosphate group is linked to the main-chain carbonyl oxygen atom of Tyr15 through an intermediate water molecule. The gamma-phosphate group interacts with the carbonyl oxygen atom and the side-chain of Asn17.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization and preliminary X-ray analysis of PepC, a thiol aminopeptidase from Lactoccocus lactis homologous to bleomycin hydrolase.

Crystals of the recombinant thiol aminopeptidase PepC, from Lactoccocus lactis, have been obtained using the hanging-drop method of vapor diffusion from ammonium sulfate solutions. Crystals are rhombohedral, the space group is R32, a = 175.2 A, c = 94.5 A (hexagonal setting). The asymmetric unit probably contains one monomer of a hexameric molecule-arrangement of 300 kDa which exhibits the crystallographic point group of symmetry 32. The crystals diffract to at least 3 A resolution.

Structural homology in the amino-terminal domains of two aminoacyl-tRNA synthetases.

The three-dimensional structures of two animoacyl-tRNA synthetases, the methionyl-tRNA synthetase from Escherichia coli (MetRS) and the tyrosyl-tRNA synthetase from Bacillus stearothermophilus (TyrRS), show a remarkable similarity over a span of about 140 amino acids. The region of homologous folding corresponds to a five-stranded parallel beta-sheet, including a mononucleotide-binding fold. One cysteine and two histidine residues that were found to be invariant in the amino acid sequences occupy similar places in the nucleotide-binding fold. In TyrRS, these residues are close to the adenylate binding site, and in MetRS to the Mg2+-ATP binding site.

Crystallization and preliminary X-ray diffraction studies of beta-cryptogein, a toxic elicitin secreted by the phytopathogenic fungus Phytophthora cryptogea.

Crystals of the basic elicitin secreted by Phytophthora cryptogea have been obtained by the hanging-drop method of vapor diffusion from sodium chloride solutions. The crystals belong to the tetragonal space group P4(1)22 (or enantiomorph P4(3)22), with unit cell dimensions a = b = 47 A, c = 137 A and probably contain two molecules per asymmetric unit. The crystals are very stable to X-rays and diffract to 2.2 A resolution on a synchrotron radiation source.

Dimeric crystal structure of a Bowman-Birk protease inhibitor from pea seeds.

The trypsin/chymotrypsin inhibitors from winter pea seeds (PsTI) are members of the Bowman-Birk protease inhibitor (BBPI) family. The crystal structure of the isoform PsTI-IVb was determined by molecular replacement at 2.7 A resolution using the X-ray co-ordinates of the soybean inhibitor as a search model. The inhibitor crystallized with a nearly perfect 2-fold symmetric dimer in the asymmetric unit. Although the overall structure is very similar to that seen in other BBPIs, there are notable new structural features. Unlike the previously reported X-ray structures of BBPIs, the structure of PsTI-IVb includes the C-terminal segment of the molecule. The C-terminal tail of each subunit is partly beta-stranded and interacts with the 2-fold symmetry-related subunit, forming a beta-sheet with strands A and B of this subunit. The dimer is mainly stabilized by a large internal hydrogen-bonded network surrounded by two hydrophobic links. Fluorescence anisotropy decay measurements show that residues Tyr59 and Tyr43 are mobile in the picosecond time scale with a large amplitude. The fluorescence study and a molecular model of the simultaneous binding of PsTI-IVb to porcine trypsin and bovine chymotrypsin are compatible only with a monomeric state of the functional molecule in solution.

The 2.1 A structure of an elicitin-ergosterol complex: a recent addition to the Sterol Carrier Protein family.

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in tobacco both remote leaf necrosis and the induction of a resistance against subsequent attack by various microorganisms. Despite the recent description of the three-dimensional crystal structure of cryptogein (CRY), the molecular basis of the interactions between Phytophthora and plants largely remains unknown. The X-ray crystal structure, refined at 2.1 A, of a ligand complexed, mutated CRY, K13H, is reported. Analysis of this structure reveals that CRY is able to encapsulate a ligand that induces only a minor conformational change in the protein structure. The ligand has been identified as an ergosterol by gas chromatographic analysis coupled with mass spectrometry analysis. This result is consistent with biochemical data that have shown that elicitins are a distinct class of Sterol Carrier Proteins (SCP). Data presented here provide the first structural description of the pertinent features of the elicitin sterol interaction and permit a reassessment of the importance of both the key residue 13 and the mobility of the omega loop for the accessibility of the sterol to the cavity. The biological implications thereof are discussed. This paper reports the first structure of a SCP/sterol complex.

Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase.

Comparison of the amino-acid sequences of several methionyl-tRNA synthetases indicates the occurrence of a few conserved motifs, having a possible functional significance. The role of one of these motifs, centered at position 300 in the E. coli enzyme sequence, was assayed by the use of site-directed mutagenesis. Substitution of the His301 or Trp305 residues by Ala resulted in a large decrease in methionine affinity, whereas the change of Val298 into Ala had only a moderate effect. The catalytic rate of the enzyme was unimpaired by these substitutions. It is concluded that the above conserved amino-acid region is located at or close to the amino-acid binding pocket of methionyl-tRNA synthetase.

Methionyl-tRNA synthetase from E. coli--a review.

Methionyl-tRNA synthetase (MetRS) from E coli is a dimer composed of 2 identical subunits of Mr 76 kDa. A fully active monomeric fragment (64 kDa) could be obtained by mild proteolysis of the native dimer. Earlier studies reviewed in Blanquet et al (1979) have compared the catalytic mechanisms of native and trypsin-modified MetRS. Moreover, the truncated form of the enzyme was crystallized and its 3-D structure solved at low resolution. In the last few years, the availability of the corresponding metG gene has facilitated the development of studies using affinity labelling and site-directed mutagenesis techniques. In parallel, the 3-D structure has been solved at a resolution of 2.5 A. These convergent approaches have allowed significant progress in the understanding of the structure-function relationships of this enzyme, and, in particular, of the rules governing the recognition of tRNA.

A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases.

The superimposable dinucleotide fold domains of MetRS, GlnRS and TyrRS define structurally equivalent amino acids which have been used to constrain the sequence alignments of the 10 class I aminoacyl-tRNA synthetases (aaRS). The conservation of those residues which have been shown to be critical in some aaRS enables to predict their location and function in the other synthetases, particularly: i) a conserved negatively-charged residue which binds the alpha-amino group of the amino acid substrate; ii) conserved residues within the inserted domain bridging the two halves of the dinucleotide-binding fold; and iii) conserved residues in the second half of the fold which bind the amino acid and ATP substrate. The alignments also indicate that the class I synthetases may be partitioned into two subgroups: a) MetRS, IleRS, LeuRS, ValRS, CysRS and ArgRS; b) GlnRS, GluRS, TyrRS and TrpRS.

Geometry of intercalation of psoralens in DNA approached by molecular mechanics.

The results of molecular mechanical calculations on intercalation complexes of 3-carbethoxypsoralen, 5-methoxypsoralen, 8-methoxypsoralen, 7-methylpyrido[3,4-c]psoralen (MepyPs) and 7-methylpyrido[4,3-c]psoralen (2N-MePyPs) with the double stranded duodecanucleotide d(CGCGATATCGCG)2 are presented. In the energy-minimized structures, the psoralens are intercalated with their plane orthogonal to the helix axis. Stacking interactions between the furan ring of the psoralen and the adjacent bases are maximized in most derivatives studied, whereas the effect of the various substituents of the psoralen ring is to specifically push part of the molecule towards either the minor or the major groove, preventing a symmetrical intercalation (with respect to the two strands of the DNA). The relative position of the psoralen ring and of the adjacent thymine foreshadows the formation of furan-side monoadducts in 3-CPs, MePyPs and 2N-MePyPs, whereas the formation of a pyrone-side monoadduct appears as geometrically more favourable in 5-MOP and both furan- and pyrone-side monoadducts can be geometrically envisaged in 8-MOP. A good correlation therefore exists between the more or less favourable equilibrium geometries and the experimentally observed photoreactions. The present study is the first attempt to characterize the geometrical parameters as part of a complex set of geometrical, dynamical and excited state parameters governing the overall DNA-psoralen photoreaction.

A possible binding path of ergosterol within elicitins revealed by molecular dynamics.

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in the tobacco plant both remote leaf necrosis and the induction of a resistance against subsequent attack by various micro-organisms. The crystal structure of b-cryptogein (CRY), secreted by Phytophthora cryptogea, was previously reported as well as the first structure of a SCP/sterol complex, the ergosterol-complexed, mutated CRY (K13H). In K13H, the ergosterol molecule is encapsulated in a large internal hydrophobic cavity which is not present in CRY. This binding induces a minor conformational change in the protein structure. Molecular dynamics studies were undertaken to precise the structural behaviour of CRY and K13H with respect to the complexation of the ergosterol. Although it is not possible to simulate the entrance of the ergosterol in the protein, we assume that capture and release of the ligand possibly both occur following the same path. Our results show that, in the complex K13H, the ergosterol molecule is pushed towards the residue 13 which play a key role in the necrotic activity of the protein. It is likely that the polarity of residue 13, favouring the binding of the hydroxyl of the ligand, would be involved in the recognition of the sterol and in an optimisation of its orientation. Thus, in a first step, the molecule of ergosterol would be rotated around itself to a position which makes possible, in a second step, its translation to the internal cavity, as a key in a keyhole.

Molecular mechanics and dynamics of DNA-furocoumarin complexes: effect of the aromatization of the pyrone ring on the intercalation geometry.

Results of molecular mechanics and dynamics calculations on intercalation complexes of DNA with various furocoumarins (psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen) and their corresponding aromatized derivatives are presented. These calculations were undertaken with the aim to elucidate the roles of the pyrone and pyridine moieties in the interactions which tend to orient the furocoumarins and pyridopsoralens between DNA base pairs. It appears that the intercalation geometries are very similar for the furocoumarins and related aromatized compounds. Therefore the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not important in the orientation of the drug within the oligonucleotide.

A unique structural feature of a phospholipase A2 is probed by molecular dynamics.

The unusual catalytic network, revealed by the crystal structure of one of the two phospholipases A2 (PLA2) from the venom of the crotalid A.p.piscivorus has been probed using molecular dynamics. The catalytic network has been remodeled to a conformation similar to that found in all other PLA2, and the modeled structure has been submitted to energy minimization and molecular dynamics simulation, to explore the conformational space of the network. The calculations have yielded a large reorganization of the catalytic network, which gets a conformation close to that of the crystal structure. These results suggest that the unusual catalytic network observed in the studied PLA2 is a structural feature of the protein and not a crystal artifact.

Molecular dynamics simulations of phospholipases A2.

An extensive molecular dynamics study of phospholipases A2 from pancreatic bovine and Crotalus atrox venom has shown that the well-conserved homologous core of the phospholipases A2, including the so called catalytic network, is very stable during the course of the calculations. The fluctuations which occur are located in segments which have significantly different three-dimensional conformations in the two phospholipases A2 studied, suggesting that a particularly stable core conformation gives rise to a large homologous family of similar three-dimensional structure. The calcium ion, which exhibits a crucial structural role in the monomeric phospholipases A2, appears not to be required to stabilize the C.atrox dimer. Moreover, the behaviour of the dimeric structure during the dynamics raises the question of a possible dissociation of the two subunits into functional monomers.

Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase.

The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced-fit mechanism for methionine activation. Primary sequence comparisons of methionine-specific enzymes from yeast and bacterial sources reveals that Tyr358 is conserved in all of the known MetRS sequences.

The refined crystal structure of dimeric phospholipase A2 at 2.5 A. Access to a shielded catalytic center.

The 2.5-A crystal structure of the calcium-free form of the dimeric venom phospholipase A2 from the Western Diamondback rattlesnake Crotalus atrox, has been refined to an R-factor of 17.8% (I greater than 2 sigma) and acceptable stereochemistry. The molecule is a nearly perfect 2-fold symmetric dimer in which most of the catalytic residues of both subunits face an internal cavity. The restricted access to the putative catalytic sites is especially puzzling as the optimal substrates for this and most other phospholipase A2 are phospholipids condensed in micellar or lamellar aggregates. We point out that substrate access to the internal cavity may be aided by calcium binding which can alter the intersubunit contacts that shield the catalytic network. We also suggest that a system of hydrogen-bonded moieties exists on the surface of the dimer that links the amino terminus to the catalytic system, through an invariant Gln 4 side chain and the backbone of the active center residue, Tyr 73. This hydrogen-bonded network is on a highly accessible surface of the dimer and would appear to contribute to the enzyme's (as opposed to the proenzyme's) special capacity to attack aggregated rather than monomeric substrate.

Dimeric character of a basic phospholipase A2 from cobra venom: experimental and modelling study.

When it is gel filtered on Sephadex in the absence of calcium ions, basic phospholipase A2 from Naja nigricollis venom elutes as a dimer. In order to study the possibility of this dimerization from a structural point of view, three-dimensional models of both monomeric and dimeric N. nigricollis phospholipases A2 have been graphically built on the basis of homologies with the phospholipases A2 from pancreatic bovine and Crotalus atrox venom. The building of a dimeric model is made possible by the deletion of a particular loop of the bovine structure. The predicted models of N. nigricollis phospholipase A2 have been checked using molecular mechanics and molecular dynamics techniques according to a suitable protocol which has been developed starting from refined X-ray structures of phospholipases A2 as the test case. The observed stability of the dimeric model, in the absence of calcium, agrees with the hypothesis of the dimerization of the basic phospholipase A2. Particularly, Arg31, which replaces the hydrophobic residue present in pancreatic bovine and C.atrox venom phospholipases A2, contributes to this stability.