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J Kuriyan

Publications and source records attributed to J Kuriyan.

At least 73 records · Page 4Linked to original sources
PubMed1993

Crystallization of DsbA, an Escherichia coli protein required for disulphide bond formation in vivo.

DsbA is a 21 kDa protein that facilitates disulphide bond formation and is required for the correct folding and stability of a number of exported proteins in Escherichia coli. Crystals of oxidized DsbA have been obtained from polyethylene glycol 8000 (20 to 25%), 0.1 M-cacodylate buffer (pH 6.5) and 1% 2-methyl-2,4-pentanediol. Oxidation of the protein is critical for reproducibly obtaining high quality crystals. The resulting crystals diffract to 2 A and belong to the monoclinic space group C2 with cell dimensions a = 117.5 A, b = 65.0 A, c76.3 A, beta = 126.3 degrees with two molecules in the asymmetric unit.

Amino Acid Sequence↗
PubMed1993

Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.

The crystal structure of the Src SH2 domain complexed with a high affinity 11-residue phosphopeptide has been determined at 2.7 A resolution by X-ray diffraction. The peptide binds in an extended conformation and makes primary interactions with the SH2 domain at six central residues: PQ(pY)EEI. The phosphotyrosine and the isoleucine are tightly bound by two well-defined pockets on the protein surface, resulting in a complex that resembles a two-pronged plug engaging a two-holed socket. The glutamate residues are in solvent-exposed environments in the vicinity of basic side chains of the SH2 domain, and the two N-terminal residues cap the phosphotyrosine-binding site. The crystal structure of Src SH2 in the absence of peptide has been determined at 2.5 A resolution, and comparison with the structure of the high affinity complex reveals only localized and relatively small changes.

Amino Acid Sequence↗
PubMed1992

Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.

Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp.

The crystal structure of the beta subunit (processivity factor) of DNA polymerase III holoenzyme has been determined at 2.5 A resolution. A dimer of the beta subunit (M(r) = 2 x 40.6 kd, 2 x 366 amino acid residues) forms a ring-shaped structure lined by 12 alpha helices that can encircle duplex DNA. The structure is highly symmetrical, with each monomer containing three domains of identical topology. The charge distribution and orientation of the helices indicate that the molecule functions by forming a tight clamp that can slide on DNA, as shown biochemically. A potential structural relationship is suggested between the beta subunit and proliferating cell nuclear antigen (PCNA, the eukaryotic polymerase delta [and epsilon] processivity factor), and the gene 45 protein of the bacteriophage T4 DNA polymerase.

Amino Acid Sequence↗
PubMed1991

X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4-A resolution.

Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidine conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. We report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from -2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4 degrees in the domains that form the site, with relative shifts of as much as 2-3 A in residue positions. These results provide a detailed view of the residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

Amino Acid Sequence↗
PubMed1991

Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction.

Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CAS registry number 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione and trypanothione, respectively. A combination of primary sequence alignments and molecular modeling, together with the high-resolution crystal structure of human glutathione reductase, identified certain residues as potentially being responsible for substrate discrimination. Site-directed mutagenesis of Escherichia coli glutathione reductase was used to test these predictions. The mutation of Asn-21 to Arg demonstrated that this single change was insufficient to generate the greater discrimination against trypanothione shown by human glutathione reductase compared with the E. coli enzyme. However, the mutation of Ala-18, Asn-21, and Arg-22 to the amino acid residues (Glu, Trp, and Asn, respectively) in corresponding positions in Trypanosoma congolense trypanothione reductase confirmed that this region of polypeptide chain is intimately involved in substrate recognition. It led to a mutant form of E. coli glutathione reductase that possessed essentially no activity with glutathione but that was able to catalyze trypanothione reduction with a kcat/Km value that was 10% of that measured for natural trypanothione reductases. These results should be of considerable importance in the design of trypanocidal drugs targeted at the differences between glutathione and trypanothione metabolism in trypanosomatids and their hosts.

Amino Acid Sequence↗
PubMed1991

Convergent evolution of similar function in two structurally divergent enzymes.

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.

Amino Acid Sequence↗
PubMed1991

Rigid protein motion as a model for crystallographic temperature factors.

The extent to which the librations of rigid molecules can model the crystallographic temperature factor profiles of proteins has been examined. For all proteins considered, including influenza virus hemagglutinin, glutathione reductase, myohemerythrin, myoglobin, and streptavidin, a simple 10-parameter model [V. Schomaker and K. N. Trueblood (1968) Acta Crystallogr. Sect. B 24, 63-76] is found to reproduce qualitatively the patterns of maxima and minima in the isotropic backbone meansquare displacements. Large deviations between the rigid molecule and individual atomic temperature factors are found to be correlated with a region in hemagglutinin for which the refined structural model is unsatisfactory and with errors in the structure in a partially incorrect model of myohemerythrin. For the high-resolution glutathione reductase structure, better results are obtained on treating each of the compact domains in the structure as independent rigid bodies. The method allows for the refinement of reliable temperature factors with the introduction of minimal parameters and may prove useful for the evaluation of models in the early stages of x-ray structure refinement. While these results by themselves do not establish the nature of the underlying displacements, the success of the rigid protein model in reproducing qualitative features of temperature factor profiles suggests that rigid body refinement results should be considered in any interpretation of crystallographic thermal parameters.

Binding Sites↗
PubMed1991

Exploration of disorder in protein structures by X-ray restrained molecular dynamics.

Conformational disorder in crystal structures of ribonuclease-A and crambin is studied by including two independent structures in least-squares optimizations against X-ray data. The optimizations are carried out by X-ray restrained molecular dynamics (simulated annealing refinement) and by conventional least-squares optimization. Starting from two identical structures, the optimizations against X-ray data lead to significant deviations between the two, with rms backbone displacements of 0.45 A for refinement of ribonuclease at 1.53 A resolution, and 0.31 A for crambin at 0.945 A. More than 15 independent X-ray restrained molecular dynamics runs have been carried out for ribonuclease, and the displacements between the resulting structures are highly reproducible for most atoms. These include residues with two or more conformations with significant dihedral angle differences and alternative hydrogen bonding, as well as groups of residues that undergo displacements that are suggestive of rigid-body librations. The crystallographic R-values obtained are approximately 13%, as compared to 15.3% for a comparable refinement with a single structure. Least-squares optimization without an intervening restrained molecular dynamics stage is sufficient to reproduce most of the observed displacements. Similar results are obtained for crambin, where the higher resolution of the X-ray data allows for refinement of unconstrained individual anisotropic temperature factors. These are shown to be correlated with the displacements in the two-structure refinements.

Hydrogen Bonding↗
PubMed1990

Preliminary crystallographic analysis of trypanothione reductase from Crithidia fasciculata.

Trypanothione reductase, a flavoprotein disulfide reductase specific to trypanosomatid parasites, has been crystallized by vapor diffusion of a protein solution (10 mg/ml) against 22% polyethylene glycol (average Mr 8000) containing 100 mM-ammonium sulfate. Crystals of a size suitable for structure determination by X-ray diffraction have been obtained by seeding protein solutions with smaller crystals. The space-group is P21 (a = 60.9 A, b = 161.8 A, c = 58.4 A, beta = 99.1 degrees). The molecular mass and volume of the unit cell suggest that there is a dimer of the enzyme in the asymmetric unit, and this is confirmed by self-rotation functions calculated using data to 4.5 A resolution. The crystals diffract to beyond 3 A resolution. Crystals of another P21 form (a = 91.3 A, b = 114.4 A, c = 92.0 A, beta = 141.3 degrees) are observed to grow under similar conditions.

Animals↗
PubMed1990

Temperature dependence of the structure and dynamics of myoglobin. A simulation approach.

The results of simulations of the structure and internal motions of carbonomonoxymyoglobin (MbCO) at two different temperatures (325 and 80 K) are presented and compared with experimental data. Properties calculated from the 120 ps trajectory at 325 K are used as a reference in the analysis of the motion of the protein at 80 K. Three separate 80 K molecular dynamics trajectories were calculated; they were started with different coordinate sets from the 325 K simulation and the lower temperature was achieved by scaling the velocities. The simulations yield results for the structural changes between 325 and 80 K that are in general accord with those from X-ray data. Both the experimental and calculated radii of gyration, distances from the center of mass and main-chain difference distance matrices show that there is a significant but inhomogeneous shrinkage with decreasing temperature. For the atomic fluctuations, by contrast, the calculated temperature dependence is very different from the X-ray results; i.e. the calculated root-mean-square backbone fluctuations decrease to 0.11 A at 80 K from 0.51 A at 325 K, while the fluctuations obtained from the X-ray B factors go from 0.56 A at 260 K to 0.47 A at 80 K. The smaller temperature dependence of the B factors suggests that there is significant conformational disorder in MbCO crystals at lower temperatures. This is in accord with the simulation results, which show that the protein is trapped in restricted regions of conformational space at 80 K, while at 325 K a much larger region is accessible to the protein. Analysis of the fluctuations at 325 K and 80 K shows that the room temperature flexibility of the protein is determined by the mobility of the loop regions and by side-chain torsional motions (in accord with earlier simulation results), while the low temperature fluctuations involve motion within a single well. Examination of the calculated iron atom fluctuations and comparison with Mossbauer data show good agreement. It is found that the dominant contribution to the iron motion arises from heme sliding; motion of the iron relative to the heme are much smaller.

Chemical Phenomena↗
PubMed1989

Crystallization and preliminary x-ray characterization of thioredoxin reductase from Escherichia coli.

Single crystals of thioredoxin reductase, suitable for x-ray diffraction studies, have been obtained at room temperature by vapor diffusion of 10-20 mg/ml protein solution against 35% polyethylene glycol containing 200 mM ammonium sulfate. Good quality crystals appear spontaneously only from a protein solution that had been stored for more than a year at 4 degrees C, although large single crystals are reproducibly obtained from fresh protein solutions by micro-seeding. The space group is P6(3)22 (a = b = 123.8 A, c = 81.6 A), with one monomer of the enzyme (34.5 kDa) in the crystallographic asymmetric unit. The crystals are well ordered and diffract to beyond 2 A resolution.

Crystallization↗
PubMed1989

X-ray refinement of protein structures by simulated annealing: test of the method on myohemerythrin.

The recently developed method of structure factor refinement by molecular dynamics with simulated annealing [Brünger, Kuriyan & Karplus (1987). Science, 235, 458-460] is tested on the 118 residue protein myohemerythrin. A highly refined structure for this protein at 1.3/1.7 A resolution has recently been published [Sheriff, Hendrickson & Smith (1987). J. Mol. Biol. 197, 273-296]. This is compared with the results of simulated annealing refinement (with no manual intervention) starting from an earlier model for the protein from a stage in the refinement when conventional least-squares methods could not improve the structure. Simulated annealing reduces the R factor at 2.5 A from 39 to 31%, with uniform temperature factors and no solvent molecules and with similar stereochemistry; the comparable value for the manually refined structure is 27.9%. Errors in backbone and sidechain positions up to about 3 A are corrected by the method. The error in backbone positions for roughly 85% of the initial structure is within this range, and in these regions the r.m.s. backbone error is reduced from 1.1 to 0.4 A. For the rest of the structure, including a region which was incorrectly built due to a sequence error, the procedure does not yield any improvement and manual intervention appears to be required. Nevertheless, the overall improvement in the structure results in electron density maps that are easier to interpret and permit identification of the errors in the structure. The general utility of the simulated annealing methodology in X-ray refinement is discussed.

Evaluation Studies as Topic↗
PubMed1987

Thermal expansion of a protein.

The thermal expansion of a protein, metmyoglobin, was investigated by analysis of the refined X-ray crystal structures at 80 and 255-300 K. On heating from 80 to 300 K, the volume occupied by myoglobin increases by approximately 3%. The linear thermal expansion coefficient is estimated to be 115 X 10(-6) K-1. This value is more than twice as large as that of liquid water but less than that of benzene. As the temperature is raised, the internal volume change does not come from the large, atom-sized internal cavities in the structure but from an increase in the small, subatomic free volumes between atoms. The largest expansion occurs in the region of the CD and GH corners; both these regions move away from the center of the protein. The remainder of the expansion results from the lengthening of contacts between segments of secondary structure.

Animals↗
PubMed1987

Estimation of uncertainties in X-ray refinement results by use of perturbed structures.

The uncertainties in the refined parameters for a 1.5-A X-ray structure of carbon-monoxy (FeII) myoglobin are estimated by combining energy minimization with least-squares refinement against the X-ray data. The energy minimizations, done without reference to the X-ray data, provide perturbed structures which are used to restart conventional X-ray refinement. The resulting refined structures have the same, or better, R-factor and stereochemical parameters as the original X-ray structure, but deviate from it by 0.13 A rms for the backbone atoms and 0.31 A rms for the sidechain atoms. Atoms interacting with a disordered sidechain, Arg 45 CD3, are observed to have larger positional uncertainties. The uncertainty in the B-factors, within the isotropic harmonic motion approximation, is estimated to be 15%. The resulting X-ray structures are more consistent with the energy parameters used in simulations.

Arginine↗