The crystal structure of prokaryotic phospholipase A2

In this study, the x-ray crystal structures of the calcium-free and calcium-bound forms of phospholipase A(2) (PLA(2)), produced extracellularly by Streptomyces violaceoruber, were determined by using the multiple isomorphous replacement and molecular replacement methods, respectively. The former and latter structures were refined to an R-factor of 18.8% at a 1.4-A resolution and an R-factor of 15.0% at a 1.6-A resolution, respectively. The overall structure of the prokaryotic PLA(2) exhibits a novel folding topology that demonstrates that it is completely distinct from those of eukaryotic PLA(2)s, which have been already determined by x-ray and NMR analyses. Furthermore, the coordination geometry of the calcium(II) ion apparently deviated from that of eukaryotic PLA(2)s. Regardless of the evolutionary divergence, the catalytic mechanism including the calcium(II) ion on secreted PLA(2) seems to be conserved between prokaryotic and eukaryotic cells. Demonstrating that the overall structure determined by x-ray analysis is almost the same as that determined by NMR analysis is useful to discuss the catalytic mechanism at the molecular level of the bacterial PLA(2).     

The first crystal structure of L-threonine dehydrogenase

L-threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.     

Crystal structure of a phospholipase D family member

The first crystal structure of a phospholipase D (PLD) family member has been determined at 2.0 A resolution. The PLD superfamily is defined by a common sequence motif, HxK(x)4D(x)6GSxN, and includes enzymes involved in signal transduction, lipid biosynthesis, endonucleases and open reading frames in pathogenic viruses and bacteria. The crystal structure suggests that residues from two sequence motifs form a single active site. A histidine residue from one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoenzyme intermediate, whereas a histidine residue from the other motif appears to function as a general acid in the cleavage of the phosphodiester bond. The structure suggests that the conserved lysine residues are involved in phosphate binding. Large-scale genomic sequencing revealed that there are many PLD family members. Our results suggest that all of these proteins may possess a common structure and catalytic mechanism.     

The crystal structure of a lyxose-bridged dimolybdate: a redetermination of the first monosaccharide-metal complex’s structure

The crystal structure of the first monosaccharide-metal complex ever reported, a β-d-lyxofuranose-bridged dimolybdate [Taylor, G. E.; Waters, J. M. Tetrahedron Lett.1981, 22, 1277-1278] was re-determined since the original work shows unbalanced charges, and thus an unclear protonation state of the lyxose ligand. As a result, the complex was found to be a monoanion whose charge is balanced by an ammonium counterion. The lyxose O5 atom is non-deprotonated, and the carbohydrate ligand is trianionic due to deprotonation of the O1, O2 and O3 hydroxy functions.     

Mammalian phospholipase D structure and regulation.

The recent identification of cDNA clones for phospholipase D1 and 2 has opened the door to new studies on its structure and regulation. PLD activity is encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiple specialized steps in receptor dependent and constitutive processes of secretion, endocytosis, and membrane biogenesis.     

The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).     

The crystal structure of a lysozyme c from housefly Musca domestica, the first structure of a digestive lysozyme

Lysozymes from family 22 of glycoside hydrolases are usually part of the defense system against bacteria. However in ruminant artiodactyls and saprophagous insects, lysozymes are involved in the digestion of bacteria. Here, we report the first crystallographic structure of a digestive lysozyme in its native and complexed forms, the structure of lysozyme 1 from Musca domestica larvae midgut (MdL1). Structural and biochemical data presented for MdL1 are analyzed in light of digestive lysozymes' traits. The structural core is similar, but a careful analysis of a structural alignment generated with other lysozymes c reveals that significant differences occur in coil regions. The loop from MdL1 defined by residues 98-100 has one deletion previous to residue Gln100, which leads to a less exposed conformation and might justify the resistance to proteolysis observed for MdL1. In addition, Gln100 is directly involved in a few hydrogen bonds to the ligand in a yet unobserved substrate binding mode. The pK(a)s of the MdL1 catalytic residues (Glu32 and Asp50) are lower (6.40 and 3.09, respectively) than those from Gallus gallus egg lysozyme (GgL, hen egg white lysozyme-HEWL) (6.61 and 3.85, respectively). A unique feature of MdL1 is a hydrogen bond between Thr107 Ogamma and Glu32 carboxylate group, which combined with the presence of Ser106 contributes to decrease the pK(a) of Glu32. Furthermore, in MdL1 the presence of Asn46 preventing the occurrence of an electrostatic repulsion with Asp50 and the increment in the solvent exposition of Asp50 due to Pro42 insertion contribute to reduce the pK(a) of Asp50. These structural elements affecting the pK(a)s of the catalytic residues should contribute to the acidic pH optimum presented by MdL1.     

The first crystal structure of a Mimosoideae lectin reveals a novel quaternary arrangement of a widespread domain

The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of beta-prism domains, and highlight the adaptability of the beta-prism fold as a building block in the evolution of plant lectins. The P.platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroid-shaped structure of the P.platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P.platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms.     

Insights into the structure of plant alpha-type phospholipase D

Phospholipases D play an important role in the regulation of cellular processes in plants and mammals. Moreover, they are an essential tool in the synthesis of phospholipids and phospholipid analogs. Knowledge of phospholipase D structures, however, is widely restricted to sequence data. The only known tertiary structure of a microbial phospholipase D cannot be generalized to eukaryotic phospholipases D. In this study, the isoenzyme form of phospholipase D from white cabbage (PLDalpha2), which is the most widely used plant phospholipase D in biocatalytic applications, has been characterized by small-angle X-ray scattering, UV-absorption, CD and fluorescence spectroscopy to yield the first insights into its secondary and tertiary structure. The structural model derived from small-angle X-ray scattering measurements reveals a barrel-shaped monomer with loosely structured tops. The far-UV CD-spectroscopic data indicate the presence of alpha-helical as well as beta-structural elements, with the latter being dominant. The fluorescence and near-UV CD spectra point to tight packing of the aromatic residues in the core of the protein. From the near-UV CD signals and activity data as a function of the calcium ion concentration, two binding events characterized by dissociation constants in the ranges of 0.1 mm and 10-20 mm can be confirmed. The stability of PLDalpha2 proved to be substantially reduced in the presence of calcium ions, with salt-induced aggregation being the main reason for irreversible inactivation.     

The first crystal structure of hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum

The extremely thermostable NAD-dependent glutamate dehydrogenase (NAD-GluDH) from Pyrobaculum islandicum, a member of the Crenarchaeota, was crystallized, and its 3D structure has been determined by X-ray diffraction methods. The homohexameric structure of Pb. islandicum glutamate dehydrogenase (Pis-GluDH) was solved and refined at a resolution of 2.9A with a crystallographic R-factor of 19.9% (Rfree 26.0%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. The secondary structural elements and catalytically important residues of the enzyme were highly conserved among the NAD(P)-dependent GluDHs from other sources. A structural comparison of Pis-GluDH with other NAD(P)-dependent GluDHs suggests that a significant difference in the alpha8-loop-alpha9 region of this enzyme is associated with its coenzyme specificity. From the analysis of the 3D structure, hydrophobic interactions between intersubunits were found to be important features for the enzyme oligomerization. It has been reported that Pis-GluDH is highly thermostable, like the GluDH of the hyperthermophilic archaeum Pyrococcus furiosus, and the increase in the intersubunit ion pair networks is responsible for the extreme thermostability of the Pc. furiosus enzyme. However, the number of intersubunit ion pairs in the Pis-GluDH molecules is much smaller than those of the Pc. furiosus GluDH. The number of hydrophobic interactions at the intersubunit interfaces were increased and responsible for the extremely high thermostability. This indicates that the major molecular strategy for high thermostability of the GluDHs may be different for each hyperthermophile.     

The first crystal structure of class III superoxide reductase from Treponema pallidum

Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl₂ and diffracted beyond 1.55 Å resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the β-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)₄(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.This work is dedicated to the memory of Prof. Frank Rusnak.Coordinates and observed structure factor amplitudes have been deposited in the Protein Data Bank under the accession code 1Y07.     

The first crystal structure of a macromolecular assembly under high pressure: CpMV at 330 MPa

The structure of cubic Cowpea mosaic virus crystals, compressed at 330 MPa in a diamond anvil cell, was refined at 2.8 A from data collected using ultrashort-wavelength (0.331 A) synchrotron radiation. With respect to the structure at atmospheric pressure, order is increased with lower Debye Waller factors and a larger number of ordered water molecules. Hydrogen-bond lengths are on average shorter and the cavity volume is strongly reduced. A tentative mechanistic explanation is given for the coexistence of disordered and ordered cubic crystals in crystallization drops and for the disorder-order transition observed in disordered crystals submitted to high pressure. Based on such explanation, it can be concluded that pressure would in general improve, albeit to a variable extent, the order in macromolecular crystals.     

The first crystal structure of a family 31 carbohydrate-binding module with affinity to beta-1,3-xylan

Here, we present the crystal structure of the family 31 carbohydrate-binding module (CBM) of beta-1,3-xylanase from Alcaligenes sp. strain XY-234 (AlcCBM31) determined at a resolution of 1.25A. The AlcCBM31 shows affinity with only beta-1,3-xylan. The AlcCBM31 molecule makes a beta-sandwich structure composed of eight beta-strands with a typical immunoglobulin fold and contains two intra-molecular disulfide bonds. The folding topology of AlcCBM31 differs from that of the large majority of other CBMs, in which eight beta-strands comprise a beta-sandwich structure with a typical jelly-roll fold. AlcCBM31 shows structural similarity with CBM structures of family 34 and family 9, which also adopt structures based on immunoglobulin folds.     

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.     

The transphosphatidylation activity of phospholipase D

Transphosphatidylation activity is a characteristic and remarkable property of phospholipase D (PLD) and has been studied in plants and mammalian tissues. This reaction is often used to confirm the properties and/or abnormalities of PLD activity. The mechanism for activating PLD transphosphatidylation seems multiple. Although significant changes of transphosphatidylation activity have been found in some pathological animal models, the biological significance of PLD transphosphatidylation remains largely unknown.     

The substrate requirements of phospholipase D

The hydrolysis rates of different diphosphates, compared with the one observed with natural phosphatidylcholine, are used to identify the molecular basis for phospholipase D (PLD) catalysis. Experimental data strongly support the idea that PLD is a rather generic phosphodiesterase with very wide substrate specificity and a net preference for lipophilic substrates. The presence of choline in the polar head is not required for activity although it improves hydrolysis efficiency. Choline esters are found to be substrates for PLD hydrolysis, but only with long chain fatty acids.     

The crystal structure of a Dcp-containing peptide

We have investigated the conformational preferences of a newly synthesized C(alpha,alpha) symmetrically disubstituted glycine, namely alpha,alpha-dicyclopropylglycine (Dcp). We report here the crystal structure of a fully protected dipeptide containing Dcp, namely Z-Dcp(1)-Dcp(2)-OCH(3). Both Dcp residues are in a folded conformation. The overall peptide structural organization corresponds to an alpha-pleated sheet conformation, similar to that observed in linear peptides made up of alternating D- and L-residues and in Z-Aib-Aib-OCH(3) (Aib: alpha,alpha-dimethylglycine). These preliminary data suggest that the Dcp could represent an alternative as molecular tool to stabilize folded conformations.     

The structure of a centrosymmetric protein crystal

Crystals of racemic rubredoxin, prepared by independent chemical synthesis of the two enantiomers, have been grown and characterized. The unit cell contains two molecules, one of each enantiomer. Examination of the intensity distribution in the diffraction pattern revealed that the crystals are centrosymmetric. This was confirmed by solution of thestructure to 2 Å resolution via molecular replacement methods. The electron density maps are of very high quality due to the fact that the phaseof each reflection must be exactly 0° or exactly 180°. These results demonstrate the feasibility of using synthetic racemic proteins to yield centrosymmetric protein crystals with electron density maps that have very low phase error and model bias. © 1993 Wiley-Liss, Inc.     

The first crystal structure of gluconolactonase important in the glucose secondary metabolic pathways

The first gluconolactonase crystal structure from bacteria has been determined to a resolution of 1.61 Å using X-ray crystallography. It belongs to the senescence marker protein 30/gluconolaconase superfamily but exhibits substrate specificity mainly toward d-glucono-δ-lactone. It forms a novel disulfide-bonded clamshell dimer comprising two doughnut-shaped six-bladed β-propeller domains, yet with an exceptionally long N-terminal subdomain forming an extra helix and four additional β-strands to enclose half of the outermost β-strands of each propeller. Extensive interactions, including H-bonds, salt bridges, disulfide bonds, and coordination bonds, along with numerous bridging water molecules, are present in the interface to institute the “top-to-top” clamshell-type dimer. Three calcium ions per subunit were observed. Two are present in the central water-filled channel, with the top one coordinated to four highly conserved amino acids and is possibly involved in substrate hydrolysis, while the bottom one is coordinated to the backbone oxygen atoms, which is possibly for stabilizing the propeller domain. One calcium ion is situated in the interface also to stabilize the dimer form. Since gluconolactonase is essential in the glucose secondary metabolic pathways leading to the synthesis of pentose, vitamin C, or “antiaging” factors, determination of its tertiary structure should help understand these important biochemical processes.