Crystal structure of an open conformation of citrate synthase from chicken heart at 2.8-A resolution

The X-ray structure of a new crystal form of chicken heart muscle citrate synthase, grown from solutions containing citrate and coenzyme A or L-malate and acetyl coenzyme A, has been determined by molecular replacement at 2.8-A resolution. The space group is P4(3) with a = 58.9 A and c = 259.2 A and contains an entire dimer of molecular weight 100,000 in the asymmetric unit. Both "closed" conformation chicken heart and "open" conformation pig heart citrate synthase models (Brookhaven Protein Data Bank designations 3CTS and 1CTS) were used in the molecular replacement solution, with crystallographic refinement being initiated with the latter. The conventional crystallographic R factor of the final refined model is 19.6% for the data between 6- and 2.8-A resolution. The model has an rms deviation from ideal values of 0.034 A for bond lengths and of 3.6 degrees for bond angles. The conformation of the enzyme is essentially identical with that of a previously determined "open" form of pig heart muscle citrate synthase which crystallizes in a different space group, with one monomer in the asymmetric unit, from either phosphate or citrate solution. The crystalline environment of each subunit of the chicken enzyme is different, yet the conformation is the same in each. The open conformation is therefore not an artifact of crystal packing or crystallization conditions and is not species dependent. Both "open" and "closed" crystal forms of the chicken heart enzyme grow from the same drop, showing that both conformations of the enzyme are present at equilibrium in solution containing reaction products or substrate analogues.     

Refinement of the structure of bovine seminal ribonuclease

We report here the refinement at 2.5-Å resolution of the x-ray crystal structure of bovine seminal ribonuclease, a dimeric covalent enzyme. The protein, which crystallizes with one molecule in the asymmetric unit, consists of two subunits of identical chemical sequences, related by an almost exact binary axis. The tertiary structure of the subunits is similar to that of the pancreatic enzyme, which shows similar catalytic properties. The refinement was carried out using the restrained least-squares procedure both in the reciprocal and real spaces. The assemblage of the subunits in the dimer is described and discussed.     

Structure of D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor in a tetragonal crystal form

D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion.The crystals have space group P4212,cell parameters a=15.49 nm,c=8.03 nm and two subunits per asymmetric unit.The crystal structure at 0.34 nm was determined by the molecular replacement method.The final model has crystallographic Rfree and R factors of 0.274 and 0.262,and r.m.s.deviations of 0.002 nm for bond lengths and 2.33°for bond angles.The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure,but also in average temperature factors.This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits.This further supports that holo-GAPDH has a good 222 molecular symmetry.     

Crystallization and preliminary X-ray analysis of pig kidney DOPA decarboxylase.

DOPA decarboxylase from pig kidney, an alpha 2 dimeric enzyme of Mr = 107,000, has been crystallized by the vapour diffusion method with ammonium sulphate as precipitant. The crystals belong to the space group P6(2) (or its enantiomer P6(4)) and have unit cell dimensions of a = b = 155.9 A, c = 87.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. They diffract to 2.6 A resolution. There is one dimeric molecule per asymmetric unit. Rotation function studies have revealed the orientation of the non-crystallographic 2-fold axis of the dimer in the asymmetric unit.     

Molecular and crystal structure of (1----3)-alpha-D-glucan triacetate.

A crystal and molecular structure for GTA I, the low temperature polymorph of (1----3)-alpha-D-glucan triacetate, is proposed on the basis of X-ray diffraction analysis of well-oriented films, combined with stereochemical model refinement. The unit cell is monoclinic with parameters a = 30.17 A, b = 17.42 A, c (fibre axis) = 12.11 A, and beta = 90 degrees C. The probable space group is P2(1) with b axis unique. Six molecular chains pass through the unit cell with alternating polarity and with three independent chains comprising the asymmetric unit. The chain axes are located in a hexagonal packing arrangement. The chain backbone conformation is a left-handed, three-fold helix, but all nine O(6) acetyl groups of the asymmetric unit are in non-equivalent rotational positions. The most probable structure is indicated by X-ray residuals R = 0.261 and R" = 0.283, based on 62 reflection intensities (41 observed and 21 unobserved).     

Crystallization of a type III chloramphenicol acetyl transferase

A type III variant of chloramphenicol acetyl transferase was purified from Escherichia coli and crystallized in the presence of cobaltic hexamine chloride and 2-methyl-2, 4-pentandiol. Two crystal forms were obtained, one of which proved to be suitable for high-resolution X-ray diffraction studies. The space group of this form is R32 with aR = 74.5 A, alpha R = 92.5 degrees, with a monomer (Mr 25,000) in the asymmetric unit. The crystals diffract to 1.7 A resolution. The crystal symmetry has led to a re-evaluation of the oligomeric symmetry of the enzyme and the proposal that it is a trimer rather than a tetramer, the quaternary structure predicted previously from studies of the association of hybrid s subunits.     

Crystal structure and magnetic properties of a tetramer with a step-like Cu4N4O2 core containing asymmetric bridged dimeric subunits related to the hemocyanin copper active site

The Cu(II)-derivate of the macrocycle L derived from 2-hydroxy-5-methylisophthalaldehyde and 3- dimethylamino-1-propylamine forms tetranuclear {[LCu₂(N₃)₃](ClO₄)₂}₂ units. The crystal structure determined by X-ray crystallography is based on a step-like Cu₄N₄O₂-core with interdimeric μ(1)-azido bridging. The tetramer is formed from two dimeric subunits which are connected by a center of symmetry. The subunits are asymmetric bridged dkneric complexes with a phenolate and μ(1)-azido bridge. The coordination polyhedra around the two copper atoms are 4 + 2 and 4 + 1 including one coordinating perchlorate ion.The magnetic susceptibility of the complex has been measured in the temperature range 4.2 K to 315.6 K. The theoretical analysis using models for tetrameric and dimeric interactions revealed only a small interdimeric interaction. The magnetic exchange coupling of the asymmetric bridged dimeric unit (2J = ?528 cm^?1) is comparable to that of known di-oxygen bridged dimers. The results are related to the magnetic behaviour of the asymmetric bridges found in the binuclear copper sites in hemocyanin.Infrared measurements have been taken and there is a discussion of the asymmetric vibrations of the three different azido-groups.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae

The bilin binding protein of the butterfly Pieris brassicae has been prepared, crystallized and its crystal structure determined at high resolution using film and FAST area detector intensity data. The crystallographic asymmetric unit contains a tetramer of identical subunits with a molecular weight of about 90,000. The crystal structure was determined by isomorphous replacement. Use was made of the molecular symmetry to improve phases. A molecular interpretation of the electron density distribution and partial tracing of the polypeptide chain was possible without amino acid sequence information, as the fold is very similar to retinol binding protein. It is characterized by a beta-barrel formed by two orthogonal beta-sheets and an alpha-helix. The bilin pigment seems to be bound within the beta-barrel analogously to retinol in retinol binding protein. The tetramer in the crystal has C2 symmetry and is a dimer of dimers of quasi-equivalent subunits.     

Biochemical characterization and crystal structure determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism

Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD+ to NADH, as part of the beta-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other alpha/beta dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central beta-sheet. The second, or C-terminal, domain is primarily alpha-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD+ complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.     

Crystallization and preliminary X-ray analysis of gamma-aminobutyric acid transaminase

gamma-Aminobutyric acid transaminase from pig liver, an alpha 2 dimeric enzyme of Mr 110,100, has been crystallized by the vapour diffusion method with polyethylene glycol as precipitant. The crystals are monoclinic, space group P2(1), unit cell dimensions a = 82.1 A, b = 230.0 A, c = 70.3 A, beta = 123.9 degrees and diffract to 2.5 A resolution. There are two dimers per asymmetric unit.     

The molecular and crystal structure of dextrans: a combined electron and X-ray diffraction study. II. A low temperature, hydrated polymorph

The crystal and molecular structure of a dextran hydrate has been determined through combined electron and X-ray diffraction analysis, aided by stereochemical model refinement. A total of 65 hk0 electron diffraction intensities were measured on frozen single crystals held at the temperature of liquid nitrogen, to a resolution limit of 1.6 A. The X-ray intensities were measured from powder patterns recorded from collections of the single crystals. The structure crystallizes in a monoclinic unit cell with parameters a = 25.71 A, b = 10.21 A, c (chain axis) = 7.76 A and beta = 91.3 degrees. The space group is P2(1) with b axis unique. The unit cell contains six chains and eight water molecules, with three chains of the same polarity and four water molecules constituting the asymmetric unit. Along the chain direction the asymmetric unit is a dimer residue; however, the individual glucopyranose residues are very nearly related by a molecular 2-fold screw axis. The conformation of the chain is very similar to that in the anhydrous structure, but the chain packing differs in the two structures in that the rotational positions of the chains about the helix axes (the chain setting angles) are considerably different. The chains still pack in the form of sheets that are separated by water molecules. The difference in the chain setting angles between the anhydrous and hydrate structures corresponds to the angle between like unit cell axes observed in the diffraction diagrams recorded from hybrid crystals containing both polymorphs. Despite some beam damage effects, the structure was determined to a satisfactory degree of agreement, with the residuals R'(electron diffraction) = 0.258 and R(X-ray) = 0.127.     

Crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase: extreme asymmetry in a tetramer of identical subunits

Phosphoglycerate dehydrogenases exist in at least three different structural motifs. The first D-3-phosphoglycerate dehydrogenase structure to be determined was from Escherichia coli and is a tetramer composed of identical subunits that contain three discernable structural domains. The crystal structure of D-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 A. This enzyme represents a second structural motif of the D-3-phosphoglycerate dehydrogenase family, one that contains an extended C-terminal region. This structure is also a tetramer of identical subunits, and the extended motif of 135 amino acids exists as a fourth structural domain. This intervening domain exerts quite a surprising characteristic to the structure by introducing significant asymmetry in the tetramer. The asymmetric unit is composed of two identical subunits that exist in two different conformations characterized by rotation of approximately 180 degrees around a hinge connecting two of the four domains. This asymmetric arrangement results in the formation of two different and distinct domain interfaces between identical domains in the asymmetric unit. As a result, the surface of the intervening domain that is exposed to solvent in one subunit is turned inward in the other subunit toward the center of the structure where it makes contact with other structural elements. Significant asymmetry is also seen at the subunit level where different conformations exist at the NAD-binding site and the putative serine-binding site in the two unique subunits.     

Structure of Helicobacter pylori catalase, with and without formic acid bound, at 1.6 A resolution

Helicobacter pylori produces one monofunctional catalase, encoded by katA (hp0875). The crystal structure of H. pylori catalase (HPC) has been determined and refined at 1.6 A with crystallographic agreement factors R and R(free) of 17.4 and 21.9%, respectively. The crystal exhibits P2(1)2(1)2 space group symmetry and contains two protein subunits in the asymmetric unit. The core structure of the HPC subunit, including the disposition of a heme b prosthetic group, is closely related to those of other catalases, although it appears to be the only clade III catalase that has been characterized that does not bind NADPH. The heme iron in one subunit of the native enzyme appears to be covalently modified, possibly with a perhydroxy or dioxygen group in a compound III-like structure. Formic acid is known to bind in the active site of catalases, promoting the breakdown of reaction intermediates compound I and compound II. The structure of an HPC crystal soaked with sodium formate at pH 5.6 has also been determined to 1.6 A (with R and R(free) values of 18.1 and 20.7%, respectively), revealing at least 36 separate formate or formic acid residues in the HPC dimer. In turn, the number of water molecules refined into the models decreased from 1016 in the native enzyme to 938 in the formate-treated enzyme. Extra density, interpreted as azide, is found in a location of both structures that involves interaction with all four subunits in the tetramer. Electron paramagnetic resonance spectra confirm that azide does not bind as a ligand of the iron and that formate does bind in the heme pocket. The stability of the formate or formic acid molecule found inside the heme distal pocket has been investigated by calculations based on density functional theory.     

Crystal structure of cambialistic superoxide dismutase from porphyromonas gingivalis.

The crystal structure of cambialistic superoxide dismutase (SOD) from Porphyromonas gingivalis, which exhibits full activity with either Fe or Mn at the active site, has been determined at 1.8-A resolution by molecular replacement and refined to a crystallographic R factor of 17.9% (Rfree 22.3%). The crystals belong to the space group P212121 (a = 75.5 A, b = 102.7 A, c = 99.6 A) with four identical subunits in the asymmetric unit. Each pair of subunits forms a compact dimer, but not a tetramer, with 222 point symmetry. Each subunit has 191 amino-acid residues most of which are visible in electron density maps, and consists of seven alpha helices and one three-stranded antiparallel beta sheet. The metal ion, a 3 : 1 mixture of Fe and Mn, is coordinated with five ligands (His27, His74, His161, Asp157, and water) arranged at the vertices of a trigonal bipyramid. Although the overall structural features, including the metal coordination geometry, are similar to those found in other single-metal containing SODs, P. gingivalis SOD more closely resembles the dimeric Fe-SODs from Escherichia coli rather than another cambialistic SOD from Propionibacterium shermanii, which itself is rather similar to other tetrameric SODs.     

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Pig heart NAD-dependent isocitrate dehydrogenase has a subunit structure consisting of alpha 2 beta gamma, with the alpha subunit exhibiting a molecular weight of 39,000 and the beta and gamma each having molecular weights of 41,000. The amino-terminal sequences (33-35 residues) and the cysteinyl peptide sequences have now been determined by using subunits separated by chromatofocusing or isoelectric focusing and electroblotting. Displacement of the N-terminal sequence of the alpha subunit by 11-12 amino acids relative to that of the larger beta and gamma subunits reveals a 17 amino acid region of great similarity in which 10 residues are identical in all three subunits. The complete enzyme has 6.0 free SH groups per average subunit of 40,000 daltons, but yields 15 distinguishable cysteines in isolated tryptic peptides. Six distinct cysteines in sequenced peptides have been located in the alpha subunit. The beta and gamma subunits contain seven and five cysteines, respectively, with tryptic peptides containing three cysteines being common to the beta and gamma subunits. The three subunits appear to be closely related, but beta and gamma are more similar to each other than either is to the alpha subunit. The NAD-specific isocitrate dehydrogenase from pig heart has been shown to have 2 binding sites/enzyme tetramer for isocitrate, manganous ion, NAD+, and the allosteric activator ADP [Colman, R. F. (1983) Pept. Protein Rev. 1, 41-69]. It is proposed that the catalytically active tetrameric enzyme is organized as a dimer of dimers in which the alpha beta and alpha gamma dimers are nonidentical but functionally similar.     

DNA knotting caused by head-on collision of transcription and replication

Obtaining crystals of membrane proteins that diffract to high resolution remains a major stumbling block in structure determination. Here we present a new method for crystallizing membrane proteins from a bicelle forming lipid/detergent mixture. The method is flexible and simple to use. As a test case, bacteriorhodopsin (bR) from Halobacterium salinarum was crystallized from a bicellar solution, yielding a new bR crystal form. The crystals belong to space group P2(1) with unit cell dimensions of a=45.0 A, b=108.9 A, c=55.9 A, beta=113.58 degrees and a dimeric asymmetric unit. The structure was solved by molecular replacement and refined at 2.0 A resolution. In all previous bR structures the protein is organized as a parallel trimer, but in the crystals grown from bicelles, the individual bR subunits are arranged in an antiparallel fashion.     

Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase.

Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells. The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees. The asymmetric unit contains either a dimeric enzyme molecule, or a monomer.     

Preliminary X-ray crystallographic study of adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase, a dimeric enzyme of 96,000 Mr, catalyzes the first committed step toward the de novo biosynthesis of AMP. Large, single crystals of adenylosuccinate synthetase from Escherichia coli grow from solutions of polyethylene glycol and ammonium sulfate. Crystals from ammonium sulfate belong to the orthorhombic space group P212121 with unit cell parameters a = 79.0 A, b = 70.2 A and c = 152.6 A. Crystals from polyethylene glycol belong to the space group P21, having unit cell parameters of a = 71.16 A, b = 71.99 A, c = 82.95 A, and beta = 71.52 degrees. The asymmetric units of both crystal forms probably contain the entire dimeric enzyme.