Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1989 Aug 20
PMID:Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes. 255 83

Linear dichroism measurements were performed in the wavelength region 250 to 350 nm on complexes between the single-stranded DNA binding protein of bacteriophage T4 (gp32) and single-stranded DNA and a variety of homopolynucleotides in compressed polyacrylamide gels. The complexes appeared to orient well, giving rise to linear dichroism spectra that showed contributions from both the protein aromatic residues and the bases of the polynucleotides. In most cases the protein contribution appeared to be very similar, and the linear dichroism of the bases could be explained by similar orientations of the bases for most of the complexes. Assuming a similar, regular structure for most of the polynucleotides in complex, only a limited set of combinations of tilt and twist angles can explain the linear dichroism spectra. These values of tilt and twist are close to (-40 degrees, 30 degrees), (-40 degrees, 150 degrees), (40 degrees, -30 degrees) or (40 degrees, -150 degrees), with an uncertainty in both angles of about 15 degrees. Although the linear dichroism results do not allow a choice between these possible orientations, the latter two combinations are not in agreement with earlier circular dichroism calculations. For the complexes formed with poly(rC) and poly(rA), the linear dichroism spectra could not be explained by the same base orientations. In these two cases also the protein contribution to the linear dichroism appeared to be different, indicating that for some aromatic residues the orientations are not the same as those in the other complexes. The different structures of these complexes are possibly related to the relatively low binding affinity of gp32 to poly(rC), and to a lesser extent to poly(rA).
J Mol Biol 1989 Oct 05
PMID:Orientation of the bases of single-stranded DNA and polynucleotides in complexes formed with the gene 32 protein of bacteriophage T4. A linear dichroism study. 258 94

The beta-sandwich in thymidylate synthase comprises two six-stranded mixed beta-sheets, each contributed by one subunit of the dimeric molecule. In contrast to other proteins of known structure in which beta-sheets stack face to face, the central beta-sheets in the thymidylate synthase dimer are related by a right-handed rather than a left-handed twist. Using a highly refined model of an Escherichia coli thymidylate synthase ternary complex, we show that the individual beta-sheets in each subunit are severely distorted by an unusual series of stacked beta-bulges, which partitions each larger sheet into two smaller beta-sheets approximately orthogonal to one another. These stacked beta-bulges are locally stabilized by hydrogen bonding involving eight conserved residues. This extended structure anchors the phosphate of bound dUMP and controls the precise orientation of the catalytically essential active site cysteine. Stereochemical factors associated with the pronounced crease caused by these stacked bulges account for the right-handed twist of opposing beta-sheets.
J Mol Biol 1989 Jan 20
PMID:Stacked beta-bulges in thymidylate synthase account for a novel right-handed rotation between opposing beta-sheets. 264 2

Rat intestinal fatty-acid-binding protein (I-FABP) is a small (15,124 Mr) cytoplasmic polypeptide that binds long-chain fatty acids in a non-covalent fashion. I-FABP is a member of a family of intracellular binding proteins that are thought to participate in the uptake, transport and/or metabolic targeting of hydrophobic ligands. The crystal structure of Escherichia coli-derived rat I-FABP with a single molecule of bound palmitate has been refined to 2 A resolution using a combination of least-squares methods, energy refinement and molecular dynamics. The combined methods resulted in a model with a crystallographic R-factor of 17.8% (7775 reflections, sigma greater than 2.0), root-mean-square bond length deviation of 0.009 A and root-mean-square bond angle deviation of 2.85 degrees. I-FABP contains ten antiparallel beta-strands organized into two approximately orthogonal, beta-sheets. The hydrocarbon tail of its single C16:0 ligand is present in a well-ordered, distinctively bent conformation. The carboxylate group of the fatty acid is located in the interior of I-FABP and forms a unique "quintet" of electrostatic interactions involving Arg106; Gln 115, and two solvent molecules. The hydrocarbon tail is bent with a slight left-handed helical twist from the carboxylate group to C-16. The bent methylene chain resides in a "cradle" formed by the side-chains of hydrophobic, mainly aromatic, amino acid residues. The refined molecular model of holo-I-FABP suggests several potential locations for entry and exiting of the fatty acid.
J Mol Biol 1989 Jul 20
PMID:Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate. 267 90

Deoxyhemoglobin S fibers associate into bundles, or fascicles, that subsequently crystallize by a process of alignment and fusion. We have used electron microscopy to study the formation of fascicles and the changes in fiber packing that occur during the conversion of fascicles to crystals. The first event in crystallization involves fibers forming fascicles that are initially small and poorly ordered but, with time, become progressively larger and more highly ordered. After six to eight hours, the fibers in a fascicle form a crystalline lattice. The three-dimensional unit cell parameters of this lattice are a = 1300 A, b = 365 A, and c = 210 A (the a axis is parallel to the fiber axis). Fibers have an elliptical cross-section whose major and minor axes are 250 A and 185 A, respectively. When projected on to the unit cell vectors, these dimensions are 210 A and 155 A, so the unit cell dimension of 365 A implies that there are two fibers per unit cell. Theoretically, fibers could pair so that each member of the unit cell is oriented in the same direction (parallel) or opposite directions (antiparallel). Fourier transforms of electron micrographs (or models) cannot distinguish between these alternatives, since the two arrangements produce very similar intensity distributions. The orientation of the fibers was determined from cross-sections of the fascicles in which the fibers are seen end-on. In this view the images of the fibers are rotationally blurred because the fibers twist 30 degrees to 40 degrees about their helical axis through the 300 A to 400 A thick section. We have been able to remove the rotational blur from each of the fibers in the unit cell using the procedures described by Carragher et al. The deblurred images of the two fibers in the unit cell are related by mirror symmetry. This relationship means that the fibers are antiparallel. These observations suggest that crystallization of fibers in fascicles is mediated by assembly of the fibers into antiparallel pairs that contain equal numbers of double strands running in each direction.
J Mol Biol 1989 Apr 20
PMID:On the assembly of sickle hemoglobin fascicles. 273 13

The 31P chemical shifts of all 13 phosphates and the chemical shifts of nearly all of the non-exchangeable protons of a symmetrical 14 base pair lac pseudooperator DNA fragment have been assigned by regiospecific labeling with oxygen-17 and two-dimensional NMR techniques. At 22 degrees C, 8 of the 13 phosphorus resonances can distinctly be resolved while the remaining 5 resonances occur in two separate overlapping regions. The 31P chemical shifts of this particular 14 base pair oligonucleotide do not follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence the more upfield the 31P resonance occurs, as shown from other 31P assignment studies. Failure of this general rule is believed to be a result of helical distortions that occur along the oligonucleotide double helix, on the basis of the analysis of Callidine [Callidine, C.R. (1982) J. Mol. Biol. 161, 343-352]. Notable exceptions to the phosphate position relationship are 5'-Py-Pu-3' dinucleotide sequences, which resonate at a lower field strength than expected in agreement with similar results as reported by Ott and Eckstein [Ott, J., & Eckstein, F. (1985) Biochemistry 24, 253]. A reasonable correlation exists between 31P chemical shifts values of the 14-mer and the helical twist sum function of Calladine. The most unusual 31P resonance occurs most upfield in the 31P spectrum, which has been assigned to the second phosphate position (5'-GpT-3') from the 5' end. This unusual chemical shift may be the result of the predicted large helical twist angle that occurs at this position in the 14-mer sequence. Further, it is believed that the large helical twist represents a unique structural feature responsible for optimum binding contact between lac repressor protein and this 14-mer lac pseudooperator segment. Assignments of proton resonances were made from two-dimensional 1H-1H nuclear Overhauser effect (NOESY) connectivities in a sequential manner applicable to right-handed B-DNA, in conjunction with two-dimensional homonuclear and heteronuclear J-correlated spectroscopies (1H-1H COSY and 31P-1H HETCOR). Most nonexchangeable base proton and deoxyribose proton (except for some unresolved H4', H5', and H5" protons) resonances were assigned.
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PMID:Assignment of phosphorus-31 and nonexchangeable proton resonances in a symmetrical 14 base pair lac pseudooperator DNA fragment. 282 Apr 76

DNA supercoiling on the nucleosome was investigated by relaxing with topoisomerase I mono- and dinucleosomes reconstituted on small DNA rings. Besides 359 base-pair (bp) rings whose linking differences were integers, two additional series of rings with fractional differences, 341 and 354 bp in size, were used. Mononucleosomes reconstituted on 359 bp rings were found to relax into a single mononucleosome form. In contrast, 341 and 354 bp mononucleosomes relaxed into a mixture of two forms, corresponding to two adjacent topoisomers. The observation that the ratio between these two forms was, within each ring series, virtually independent of the initial linking number of the topoisomer used for the reconstitution suggested that each partition reflected an equilibrium. Comparison with the equilibria observed for the same rings in the absence of histones showed that the formation of a single nucleosome is associated with a linking number change of -1.1(+/-0.1) turn. Dinucleosomes, in contrast, were not relaxed to completion and do not reach equilibria. The corresponding linking number change per nucleosome was, however, estimated to be similar to the above figure, in agreement with previous data from the literature obtained with circular chromatins containing larger numbers of nucleosomes. DNA structure in mononucleosomes was subsequently investigated by means of high-resolution electron microscopy and gel electrophoresis. It was found that the above linking number reduction could be ascribed to a particle with a large open extranucleosomal DNA loop and with no more than 1.5 turns of a superhelix around the histone core. A theoretical model of a nucleosome on a small ring was constructed in which one part of the DNA was wrapped around a cylinder and the other part was free to vary both in torsion and flexion. The linking number reduction predicted was found to be most consistent with experimental data when the twist of the DNA in the superhelix was between 10.5 and 10.65 pb per turn, suggesting that wrapping on the nucleosome does not alter the twist of the DNA significantly. A lower estimate of the linking number reduction associated with a two-turn nucleosome was also derived, based on an analysis of recent data obtained upon treatment of reconstituted minichromosomes with gyrase. The value, 1.6 turns, set a lower limit of 10.44 bp per turn for the twist of nucleosomal DNA, in agreement with the above estimate.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1988 Mar 20
PMID:Chromatin reconstitution on small DNA rings. II. DNA supercoiling on the nucleosome. 283 95

We describe a pseudo-atomic model of supercoiled DNA. Each base-pair of the DNA is represented in the model by three particles placed in a plane. The particle triplets are stacked to model stacked base-pairs in double-helical DNA, and closed circular conformations are generated to investigate supercoiling. This model is less detailed than all-atom models, which are too computationally demanding to be used to study supercoiling. On the other hand, this model contains details at the base-pair level and is therefore more elaborate than elastomechanical models. A potential energy function is written in terms of a set of internal co-ordinates defined to resemble a limited number of helical parameters. The modeled helical parameters, helical twist, base-roll, tilt and rise, are the most important parameters of the global shape of DNA. Experimentally measured mechanical properties of DNA are used to define the forces holding the particles together. We then use a procedure incorporating energy minimization and molecular dynamics to locate low energy conformations of the model DNA. The model was found to behave very much like rubber-tubing and elastomechanical models. The conformations and the effects of supercoiling pressure (a number proportional to the degree to which the total twist of the DNA has been altered from its natural value) on these conformations are all very similar to those observed in the latter two models. We also used this model to examine the effects of supercoiling pressure, base-sequence and mechanical properties on the conformations and energies of five sequences. The sequences studied include models of naturally straight DNA and DNA with static or natural bends.
J Mol Biol 1989 Feb 05
PMID:Molecular mechanics model of supercoiled DNA. 292 19

A restrained least-squares refinement of the solution structure of the self-complementary B DNA hexamer 5'd(C-G-T-A-C-G)2 is presented. The structure is refined on the basis of 190 inter-proton distances determined by pre-steady-state nuclear Overhauser enhancement measurements. Two refinements were carried out starting from two initial B DNA structures differing by an overall root-mean-square (r.m.s.) difference of 0.32 A. In both cases, the final r.m.s. difference between the experimental and calculated inter-proton distances was 0.12 A compared to 0.61 A and 0.58 A for the two initial structures. The difference between the two refined structures is small, with an overall r.m.s. difference of 0.16 A, and represents the error in the refined co-ordinates. The refined structures have a B-type conformation with local structural variations in backbone and glycosidic bond torsion angles, and base-pair propellor twist, base roll, base tilt and local helical twist angles.
J Mol Biol 1985 Sep 05
PMID:Refinement of the solution structure of the B DNA hexamer 5'd(C-G-T-A-C-G)2 on the basis of inter-proton distance data. 299 86

We have studied the structure adopted by an (A-T)34 sequence from a Xenopus globin gene when present in a negatively supercoiled plasmid. A variety of enzyme and chemical probing experiments and electrophoretic migration shift methods reveal that the sequence adopts cruciform geometry at moderate levels of supercoiling. The structure has the lowest free energy of formation yet observed for a cruciform, and no detectable kinetic barrier preventing rapid interconversion between extruded and unextruded conformations. Analysis of band-shift experiments reveals a twist change on cruciform formation of -5.8, slightly smaller than the -6.5 we would predict on the basis of a transition from B DNA. An attractive explanation consistent with this discrepancy is that the (A-T)34 stretch is locally underwound to about 11.7 base-pairs/helical turn at low levels of supercoiling. This calculation is made on the assumption that the cruciform junction is structurally similar to those examined previously, which is supported by the nuclease digestion results. This perturbed helical structure could be of considerable biological significance.
J Mol Biol 1985 Oct 05
PMID:Facile cruciform formation by an (A-T)34 sequence from a Xenopus globin gene. 299 51


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