Crystal structure of RNase A complexed with d(pA)4

Co-crystals of pancreatic RNase A complexed with oligomers of d(pA)4 were grown from polyethylene glycol 4000 at low ionic strength and the X-ray diffraction data were collected to 2.5 A resolution. From a series of heavy-atom derivatives a multiple isomorphous replacement-phased electron density map of the RNase-d(pA)4 complex was calculated to 3.5 A. By inspection, the disposition of the known structure of RNase in the unit cell was determined and this was confirmed by calculation of a standard crystallographic residual, R. Refinement of the protein alone in the unit cell as a strictly rigid body yielded an R factor of 0.32 at 2.8 A resolution. From difference Fourier syntheses DNA fragments were elucidated and incorporated into a model of the complex. The entire asymmetric unit was refined using a restrained-constrained least-squares procedure (CORELS) interspersed with difference Fourier syntheses. At the present time the crystal structure has been refined to an overall R value of 0.215 at 2.5 A resolution. The asymmetric unit of the complex crystals contains four oligomers of d(pA)4 associated with each molecule of RNase. In addition, there may also be partially ordered fragments of DNA at low occupancy present in the unit cell, but these have not, at this time, been incorporated into the model. One tetramer of d(pA)4 is entirely bound by a single protein molecule and occupies a portion of the active site cleft, filling the purine binding site and the phosphate site at the catalytic center with its 5' nucleotide. Two other tetramers are partly intermolecular. One passes from near the pyrimidine binding site over the surface of the protein toward arginine 39 and into a solvent region. A third tetramer is anchored at its 5' terminus by a salt link to lysine 98, passes near arginine and then through a solvent region to terminate with its 3' end near the surface of another protein molecule in the lattice. The fourth tetramer of d(pA)4 is bound at its 5' end on the opposite side of the protein from the active site in an electropositive anion trap that includes lysines 31 and 91 as well as arginine 33. There may be a DNA-DNA interaction involving the 5' phosphate of one tetramer and the 3' bases of two other tetramers and this may help to stabilize the crystalline complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The crystal structure of ribonuclease B at 2.5-A resolution

The glycosylated form of bovine pancreatic ribonuclease, RNase B, was crystallized from polyethylene glycol 4000 at low ionic strength in space group C2 with unit cell dimensions of a = 101.81 A, b = 33.36 A, c = 73.60 A, and beta = 90.4 degrees. The crystals, which contained two independent molecules of RNase B as the asymmetric unit, were solved by a combination of multiple isomorphous replacement and molecular replacement approaches. The structures of the two molecules were refined to 2.5-A resolution and a conventional R factor of 0.22 using a constrained-restrained least squares procedure (CORELS). Complexes were also investigated of RNase B plus ruthenium pentaamine and between RNase B and a substrate analogue iodouridine. The polypeptide backbones of the two molecules of RNase B in the asymmetric unit were found to be statistically identical and their differences from RNase A to be statistically insignificant. The carbohydrate chains of both molecules extended into solvent cavities in the crystal lattice and appear to be disordered for the most part. The oligosaccharides appear to exert no influence on the structure of the protein. Iodouridine was observed to bind identically in the pyrimidine site of both RNase B molecules and in a way apparently the same as that previously observed for RNase A. Ruthenium pentaamine bound at histidine 105 of both RNase B molecules in the asymmetric unit, but at a number of secondary sites as well. An array of bound ions was observed by Fo-Fc difference Fourier syntheses. These ions were proximal to lysine and arginine residues at the surface of the proteins while a pair of strong ion binding sites were seen to fall exactly in the active site clefts of both RNase B molecules in the asymmetric unit.     

Crystals of a catalytically defective, semisynthetic ribonuclease isomorphous with those of the fully active parent enzyme

The enzymically active, semisynthetic, non-covalent complex formed by residues 1 through 118 and residues 111 through 124 of bovine pancreatic ribonuclease A crystallizes at pH 5.2 from (NH4)2SO4/CsCl solution with space group P3(2)21 and unit cell dimensions a and b = 67.7 A, c = 65.1 A and gamma = 120 degrees. The catalytically defective enzyme that results from the replacement of phenylalanine 120 by leucine crystallizes isomorphously with the parent structure (a and b = 67.2 A, c = 64.7 A, gamma = 120 degrees).     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

Crystallization and initial crystallographic results for pepstatin A inhibited bovine cathepsin D.

Cathepsin D was purified from bovine liver by a method using two pepstatin A affinity columns. The eluted protein was combined with pepstatin A and the complex crystallized from 15% polyethylene glycol 8000 at pH 5.9. The crystals diffract to a resolution of 3.0 A and have space group P2(1)2(1)2(1) with unit cell dimensions a = 74.8 A, b = 76.0 A, c = 157.7 A. There are two molecules in the asymmetric unit. The structure was solved by molecular replacement using a pepsin search model and both molecules showed clearly interpretable density in the position expected for pepstatin A in a preliminary difference map. The refined model has r.m.s. deviations from ideal bond lengths and angles of 0.014 A and 3.2 degrees, respectively, and a crystallographic R factor of 17%.     

Atomic resolution (0.98 A) structure of eosinophil-derived neurotoxin

Human eosinophil-derived neurotoxin (EDN) is a small, basic protein that belongs to the ribonuclease A superfamily. EDN displays antiviral activity and causes the neurotoxic Gordon phenomenon when injected into rabbits. Although EDN and ribonuclease A have appreciable structural similarity and a conserved catalytic triad, their peripheral substrate-binding sites are not conserved. The crystal structure of recombinant EDN (rEDN) has been determined at 0.98 A resolution from data collected at a low temperature (100 K). We have refined the crystallographic model of the structure using anisotropic displacement parameters to a conventional R-factor of 0.116. This represents the highest resolution structure of rEDN determined to date and is only the second ribonuclease structure to be determined at a resolution greater than 1.0 A. The structure provides a detailed picture of the conformational freedom at the various subsites of rEDN, and the water structure accounts for more than 50% of the total solvent content of the unit cell. This information will be crucial for the design of tight-binding inhibitors to restrain the ribonucleolytic activity of rEDN.     

Minor groove binding of a bis-quaternary ammonium compound: the crystal structure of SN 7167 bound to d(CGCGAATTCGCG)2.

The X-ray crystal structure of the complex between the synthetic antitumour and antiviral DNA binding ligand SN 7167 and the DNA oligonucleotide d(CGCGAATTCGCG)2 has been determined to an R factor of 18.3% at 2.6 A resolution. The ligand is located within the minor groove and covers almost 6 bp with the 1-methylpyridinium ring extending as far as the C9-G16 base pair and the 1-methylquinolinium ring lying between the G4-C21 and A5-T20 base pairs. The ligand interacts only weakly with the DNA, as evidenced by long range contacts and shallow penetration into the groove. This structure is compared with that of the complex between the parent compound SN 6999 and the alkylated DNA sequence d(CGC[e6G]AATTCGCG)2. There are significant differences between the two structures in the extent of DNA bending, ligand conformation and groove binding.     

Physico-chemical basis of RecA nucleo-protein filament formation on single

The analysis of RecA protein playing a central role in homologous recombination of E. coli with single-stranded DNAs of various structure and length on quantitative level is carried out for the first time. It was shown that weak additive interactions between protein monomers of filament and different structural elements of DNA provide DNA recognition. Orthophosphate and dNMPs (I50 = 12-20 mM) were shown to be the minimal inhibitors of RecA filamentation on d(pN)20. The lengthening of homooligonucleotides from d(pN)2 to d(pN)20 by one unit leads to monotonic increase in the affinity by a factor approximately 2 (factor f) due to weak additive contacts of RecA with every internucleoside phosphate group of DNA (f = 1.56) and specific interactions with each of T and C bases (f = 1.32). RecA filament does not practically interact with bases of d(pA)n, but contacts with internucleoside phosphate groups of the first turn (n < 10; f = 2.1) more effective than with additional turns of d(pA)n (n > 10; f = 1.3). The affinity of RecA protein for d(pN)n, containing typical and a number of different modified bases depends on a type of base, peculiarities of DNA structure and conformation of its sugar-phosphate backbone. The affinity is increased significantly if the bases contain exocyclic proton accepting groups. The possible reasons of preferable complexation of RecA with DNA of definite structure and length are analyzed. The mechanism of single-stranded DNA recognition by RecA and hypothetical mechanism of homological DNA strands exchange are proposed.     

Crystal structure at 1.5-A resolution of d(CGCICICG), an octanucleotide containing inosine, and its comparison with d(CGCG) and d(CGCGCG) structures.

The octadeoxyribonucleotide d(CGCICICG) has been crystallized in space group P(6)5(22) with unit cell dimensions of a = b = 31.0 A and c = 43.7 A, and X-ray diffraction data have been collected to 1.5-A resolution. Precession photographs and the self-Patterson function indicate that 12 base pairs of Z-conformation DNA stack along the c-axis, and the double helices pack in a hexagonal array similar to that seen in other crystals of Z-DNA. The structure has been solved by both Patterson deconvolution and molecular replacement methods and refined in space group P(6)5 to an R factor of 0.225 using 2503 unique reflections greater than 3.0 sigma (F). Comparison of the molecules within the hexagonal lattice with highly refined crystal structures of other Z-DNA reveals only minor conformational differences, most notably in the pucker of the deoxyribose of the purine residues. The DNA has multiple occupancy of C:I and C:G base pairs, and C:I base pairs adopt a conformation similar to that of C:G base pairs.     

Crystal structure analysis of an A-DNA fragment at 1.8 A resolution: d(GCCCGGGC).

Single crystals of the self-complementary octadeoxyribonucleotide d(GCCCGGGC) have been analysed by X-ray diffraction methods at a resolution of 1.8 A. The tetragonal unit cell of space group P4(3)2(1)2 has dimensions of a = 43.25 A and c = 24.61 A and contains eight strands of the oligonucleotide. The structure was refined by standard crystallographic techniques to an R factor of 17.1% using 1359 3 sigma structure factor observations. Two strands of the oligonucleotide are related by the crystallographic dyad axis to form a DNA helix in the A conformation. The d(GCCCGGGC) helix is characterized by a wide open major groove, a near perpendicular orientation of base pairs to the helix axis and an unusually small average helix twist angle of 31.3 degrees indicating a slightly underwound helix with 11.5 base pairs per turn. Extensive cross-strand stacking between guanine bases at the central cytosine-guanine step is made possible by a number of local conformational adjustments including a fully extended sugar-phosphate backbone of the central guanosine nucleotide.     

Tyrosyl-base-phenylalanyl intercalation in gene 5 protein-DNA complexes: proton nuclear magnetic resonance of selectively deuterated gene 5 protein.

The interactions of oligodeoxynucleotides with the aromatic residues of gene 5 protein in complexes with d(pA)8 and d(pT)8 have been determined by 1H NMR of the protein in which the five tyrosyl residues have been selectively deuterated either in the 2,6 or the 3,5 positions. Only the 3,5 protons of the three surface tyrosyls (26, 41, and 56) interact with the bases. The remainder of the aromatic protons undergoing base-dependent upfield ring-current shifts on complex formation are phenylalanyl protons, assigned to Phe(13) on the basis of model building. 19F NMR of the complexes of the m-fluorotyrosyl-labeled protein with d(pT)4 and d(pA)8 confirms the presence of ring-current perturbations of nuclei at the 3,5-tyrosyl positions of the three surface tyrosyls. Differential expression of the 19F(1H) nuclear Overhauser effect confirms the presence of two buried and three surface tyrosyl residues. A new model of the DNA binding groove is presented involving Tyr(26)-base-Phe(13) intercalation.     

Crystal structure of a light-harvesting protein C-phycocyanin from Spirulina platensis

The crystal structure of C-phycocyanin, a light-harvesting phycobiliprotein from cyanobacteria (blue-green algae) Spirulina platensis has been solved by molecular replacement technique. The crystals belong to space group P2(1) with cell parameters a = 107.20, b = 115.40, c = 183.04 A; beta = 90.2 degrees. The structure has been refined to a crystallographic R factor of 19.2% (R(free) = 23.9%) using the X-ray diffraction data extending up to 2.2 A resolution. The asymmetric unit of the crystal cell consists of two (alphabeta)6-hexamers, each hexamer being the functional unit in the native antenna rod of cyanobacteria. The molecular structure resembles that of other reported C-phycocyanins. However, the unique form of aggregation of two (alphabeta)6-hexamers in the crystal asymmetric unit, suggests additional pathways of energy transfer in lateral direction between the adjacent hexamers involving beta155 phycocyanobilin chromophores.     

Crystallization and preliminary X-ray structure analysis of pigeon egg-white lysozyme.

Calcium binding lysozyme from pigeon egg-white was crystallized by the hanging drop vapor diffusion technique using ammonium sulphate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), and have unit cell dimensions of a = 34.2 A, b = 34.8 A, and c = 99.4 A. One asymmetric unit contains one molecule of the pigeon lysozyme. The crystals diffract X-rays at least to 2.0 A resolution and are suitable for high resolution structure analysis. The diffraction data up to 3.0 A resolution were collected with a diffraction image processor, DIP100, using a Fuji imaging plate as an area detector. The structure was solved by the molecular replacement technique and refined to an R factor of 0.216. Least-squares fitting of the main-chains of pigeon egg-white lysozyme with those of chicken egg-white lysozyme and baboon alpha-lactalbumin showed that the main-chain folding of pigeon lysozyme is more similar to that of chicken lysozyme than that of alpha-lactalbumin. The largest differences between the pigeon and chicken lysozymes are in the surface loop regions.     

X-ray crystal structure of the bovine alpha-chymotrypsin/eglin c complex at 2.6 A resolution

The crystal structure of the molecular complex formed by bovine alpha-chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 A resolution were employed to determine the structure by molecular replacement techniques. Bovine alpha-chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 A resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine alpha-chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine alpha-chymotrypsin between subsites P3 and P'2; Leu 451 acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory-inhibitors are found around the scissile peptide bond.     

Three-dimensional structure of Gln25-ribonuclease T1 at 1.84-A resolution: structural variations at the base recognition and catalytic sites.

The structure of the Gln25 variant of ribonuclease T1 (RNase T1) crystallized at pH 7 and at high ionic strength has been solved by molecular replacement using the coordinates of the Lys25-RNase T1/2'-guanylic acid (2'GMP) complex at pH 5 [Arni et al. (1988) J. Biol. Chem. 263, 15358-15368] and refined by energy minimization and stereochemically restrained least-squares minimization to a crystallographic R-factor of 14.4% at 1.84-A resolution. The asymmetric unit contains three molecules, and the final model consists of 2302 protein atoms, 3 sulfates (at the catalytic sites), and 179 solvent water molecules. The estimated root mean square (rms) error in the coordinates is 0.15 A, and the rms deviation from ideality is 0.018 A for bond lengths and 1.8 degrees for bond angles. Significant differences are observed between the three molecules in the asymmetric unit at the base recognition and catalytic sites.     

Crystal structure disposition of thymidylic acid tetramer in complex with ribonuclease A.

The crystal structure of ribonuclease A with bound thymidylic acid tetramer is reported at 2.5-A resolution. The diffusion of the tetramer into native orthorhombic crystals of the ribonuclease allows for the formation of a structurally stable complex where the single-stranded nucleic acid enters and leaves the enzyme's catalytic region in a persistent 5'-3' direction. The binding of the tetramer to the enzyme's surface is facilitated and mediated by electrostatic interactions between basic protein residues and nucleotide phosphates. Two pyrimidine nucleotides are bound to the enzyme's active site in a manner similar to that observed for other complexes between ribonuclease A and nucleic acid oligomers.     

Crystal structure of a barnase-d(GpC) complex at 1.9 A resolution

The ribonuclease excreted by Bacillus amyloliquefaciens, Barnase, was co-crystallized with the deoxy-dinucleotide d(GpC). The crystal structure was determined by molecular replacement from a model of free Barnase previously derived by Mauguen et al. Refinement was carried out using data to 1.9 A resolution. The final model, which has a crystallographic R factor of 22%, includes 869 protein atoms, 38 atoms from d(GpC), a sulfate ion and 73 water molecules. Only minor differences from free Barnase are seen in the protein moiety, the root-mean-square C alpha movement being 0.45 A. The dinucleotide has a folded conformation. It is located near the active site of the enzyme, but outside the protein molecule and making crystal packing contacts with neighboring molecules. The guanine base is stacked on the imidazole ring of active site His102, rather than binding to the so-called recognition loop as it does in other complexes of guanine nucleotides with microbial nucleases. The deoxyguanosine is syn, with the sugar ring in C-2'-endo conformation; the deoxycytidine is anti and C-4'-exo. In addition to the stacking interaction, His102 hydrogen bonds to the free 5' hydroxyl, which is located near the position where the 3' phosphate group is found in other inhibitors of microbial ribonucleases. While the mode of binding observed with d(GpC) and Barnase would be non-productive for a dinucleotide substrate, it may define a site for the nucleotide product on the 3' side of the hydrolyzed bond.     

A trigonal form of the idarubicin:d(CGATCG) complex; crystal and molecular structure at 2.0 A resolution.

The X-ray crystal structure of the complex between the anthracycline idarubicin and d(CGATCG) has been solved by molecular replacement and refined to a resolution of 2.0 A. The final R-factor is 0.19 for 3768 reflections with Fo > or = 2 sigma (Fo). The complex crystallizes in the trigonal space group P31 with unit cell parameters a = b = 52.996(4), c = 33.065(2) A, alpha = beta = 90 degree, gamma = 120 degree. The asymmetric unit consists of two duplexes, each one being complexed with two idarubicin drugs intercalated at the CpG steps, one spermine and 160 water molecules. The molecular packing underlines major groove-major groove interactions between neighbouring helices, and an unusually low value of the occupied fraction of the unit cell due to a large solvent channel of approximately 30 A diameter. This is the first trigonal crystal form of a DNA-anthracycline complex. The structure is compared with the previously reported structure of the same complex crystallizing in a tetragonal form. The geometry of both the double helices and the intercalation site are conserved as are the intramolecular interactions despite the different crystal forms.     

The structure of a DNA unwinding protein and its complexes with oligodeoxynucleotides by x-ray diffraction.

The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been solved to 2.3 A resolution by x-ray diffraction techniques. The molecule contains an extensive cleft region that we have identified as the DNA binding site on the basis of the residues that comprise its surface. The interior of the groove has a rather large number of basic amino acid residues that serve to draw the polynucleotide backbone into the cleft. Arrayed along the external edges of the groove are a number of aromatic amino acid side groups that are in position to stack upon the bases of the DNA and fix it in place. The cleft then acts as an elongated pair of jaws that draws the DNA between them by charge interactions involving the phosphates with the interior lysines and arginines. The jaws then close on the DNA strand through small conformation changes and the rotation of aromatic side-chains into position to stack upon the purines and pyrimidines. Complexes of the gene 5 protein with a variety of oligodeoxynucleotides have been formed and crystallized for x-ray diffraction analysis. The crystallographic parameters of four different unit cells indicate that the fundamental unit of the complex is composed of six gene 5 protein dimers. We believe this aggregate has 622 point group symmetry and is a ring formed by end to end closure of a linear array of six dimers. From our results we have proposed a double helical model for the gene 5 protein-DNA complex in which the protein forms a spindle or core around which the DNA is spooled. 5.0-A x-ray diffraction data from one of the crystalline complexes is currently being analyzed by molecular replacement techniques to obtain what we believe will be the first direct visualization of a protein-deoxyribonucleic acid complex approaching atomic resolution.