Crystallization and preliminary crystallographic studies of the decadeoxyoligonucleotide d(CpGpTpApCpGpTpApCpG)

Crystals of the self-complementary decadeoxyoligonucleotide d(CpGpTpApCpGpTpApCpG) have been grown from a solution containing [Co(NH3)6]Cl3 and spermine. The amber-colored crystals are hexagonal and belong to the space group P6(5) (or P6(1] with unit cell parameters a = 17.93 A, c = 43.41 A. Precession photography and molecular packing considerations indicate that the unit cell consists of a 12 nucleotide duplex. The asymmetric unit, therefore, is a disordered duplex dimer in which each pyrimidine-purine base-pair is occupied 60% of the time by a C . G pair and 40% of the time by a T . A pair. The above considerations and preliminary structure analysis reveal that this alternating pyrimidine-purine oligomer assumes a Z-DNA conformation.     

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.     

Crystal structure of the self-complementary 5''-purine start decamer d(GCGCGCGCGC) in the Z-DNA conformation. I.

Alternating self-complementary oligonucleotides starting with a 5'-pyrimidine usually form left-handed Z-DNA; however, with a 5'-purine start sequence they form the right-handed A-DNA. Here we report the crystal structure of the decamer d(GCGCGCGCGC) with a 5'-purine start in the Z-DNA form. The decamer crystallizes in the hexagonal space group P6(5)22, unit cell dimensions a = b = 18.08 and c = 43.10 A, with one of the following four dinucleotide diphosphates in the asymmetric unit: d(pGpC)/d(GpCp)/d(pCpG)/d(CpGp). The molecular replacement method, starting with d(pGpC) of the isomorphous Z-DNA hexamer d(araC-dG)3 without the 2'-OH group of arabinose, was used in the structure analysis. The method gave the solution only after the sugar-phosphate conformation of the GpC step was manipulated. The refinement converged to a final R value of 18.6% for 340 unique reflections in the resolution range 8.0-1.9 A. A result of the sequence alternation is the alternation in the nucleotide conformation; guanosine is C3'-endo, syn, and cytidine is C2'-endo, anti. The CpG step phosphodiester conformation is the same as ZI or ZII, whereas that of the GpC step phosphodiester is "intermediate" in the sense that zeta (O3'-P bond) is the same as ZII but alpha (P-O5' bond) is the same as ZI. The duplexes generated from the dinucleotide asymmetric unit are stacked one on top of the other in the crystal to form an infinite pseudocontinuous helix. This renders it a quasi-polymerlike structure that has assumed the Z-DNA conformation further strengthened by the long inner Z-forming stretch d(CG)4. An interesting feature of the structure is the presence of water strings in both the major and the minor grooves. In the minor groove the cytosine carbonyl oxygen atoms of the GpC and CpG steps are cross-bridged by water molecules that are not themselves hydrogen bonded but are enclosed by the water rings in the mouth of the minor groove. In the major groove three independent water molecules form a zigzagging continuous water string that runs throughout the duplex.     

Hexagonal crystal structure of the A-DNA octamer d(GTGTACAC) and its comparison with the tetragonal structure: correlated variations in helical parameters

The alternating DNA octamer d(GTGTACAC) has been grown in a novel hexagonal crystal form. The structure has been determined and refined to a 2-A resolution, with 51 water molecules. The A-DNA conformation is a variant of that observed for the tetragonal form of the same sequence (Jain et al., 1989) containing a bound spermine. The crystals belong to the space group P6(1)22, a = b = 32.40 A and c = 79.25 A, with one strand in the asymmetric unit. The new hexagonal structure was solved by rotation and translation searches in direct space and refined to a final R value of 12.7% by using 1561 unique reflections greater than 1.5 sigma (I). The electron density clearly shows that the penultimate A7 sugar had flipped into the alternative C2'-endo pucker. This dent in the molecule can be attributed to close intermolecular contacts. In contrast, in the tetragonal structure, the DNA is distorted in the central TA step, where the A5 backbone bonds C4'-C5' and O5'-P assume trans conformations. The hexagonal double helix more closely resembles the fiber diffraction A-DNA, compared to the tetragonal form. For instance, the tilt angle is higher (16 degrees vs 10 degrees), which is correlated with a larger displacement from the helix axis (3.5 vs 3.3), a lower rise per residue (2.9 vs 3.2), and a smaller major-groove width (6.1 vs 8.7), thus indicating that the variations in these global helical parameters are correlated. The propeller twist angles in both forms are higher for the G-C base pairs (15.3 degrees, 12.14 degrees) than for the A-T base pairs (10.8 degrees, 9.1 degrees), which is the reverse of the expected order. Unlike the tetragonal structure, the hexagonal crystal structure interestingly does not contain a bound spermine molecule. Our analysis reveals that the conformational differences between the tetragonal and hexagonal forms are not entirely due to the spermine binding, and crystal packing seems to play an important role.     

Conformational analysis of arabinonucleosides and nucleotides. A comparison with the ribonucleosides and nucleotides.

Conformations of arabino nucleosides and nucleotides have been analyzed by semiempirical energy calculations. It is found that the change in the configuration of the O(2')-hydroxyl group in arabinoses compared to riboses exerts significant influence on the conformational priorities of the glycosyl and the exocyclic C(4')-C(5') bond torsions. While the anti conformations for the bases are preferred, the anti in equilibrium or formed from syn interconversion is considerably hampered compared to ribosides due to large energy barrier. Further the preferred anti glycosyl torsions are shifted to higher values for C(3')-endo puckers and in ribosides. While the gauche+ conformation around the C(4')-C(5') bond is favored for C(3')-endo arabinosides, it is strongly stabilized for C(2')-endo arabinosides only in the presence of the intrasugar hydrogen bond O(2')-H ... O(5'). The net attractive electrostatic interactions between the phosphate and the base stabilizes the preferred conformations of 5'-arabinonucleotides also.     

The X-ray crystal structure of 6-deoxy-α-l-sorbofuranose. Conformational analysis of ketohexofuranosides and their comparison with ribofuranosides and arabinofuranosides

The crystal and molecular structure of 6-deoxy-l-sorbose have been determined by the application of multisolution methods and refined to an R-index of 0.063 for 560 reflections, using three-dimensional intensity data collected on a Picker automatic diffractometer. The compound crystallizes in the space group P2₁2₁2₁ with unit-cell dimensions a = 18.470 (10), b = 7.636 (10), and c = 5.371 (8) Å; Z = 4. The molecule occurs as the α-furanose form, which is also the preponderant tautomer in solution. The puckering of the furanoid ring is C-3′-exo-C-4′-endo (₃T⁴) [equivalent to C-2′-exo-C-3′-endo (₂T³) in the numbering for d-ribose], with P and τ_m angles of -6.5 and 42.7° respectively. Conformational analysis of the known ketofuranosides shows that the ₃T⁴ (₂T³ in d-ribose numbering) puckering mode, which is typical of α-nucleosides, is favored, in contrast to the favored ³T₂ or ²T₃ puckering mode for the β-d-ribonucleosides and β-d-arabinonucleosides. The conformational differences among furanoid rings are mainly influenced by the configuration at the anomeric carbon atom. The favored orientation about the C-2′-C-1′ bond (O-5′-C-2′-C-1′-O-1′)of the ketofuranosidesis — gauche. All four hydroxyl groups are involved in donor-acceptor hydrogen bonding, and O-4′-8 appears to be involved in a bifurcated hydrogen bond to O-2′ and O-3′ of neighboring molecules.     

Structure of transfer RNA molecules containing the long variable loop

A structure is proposed for the type II tRNA molecules containing the long variable loop and the tertiary base interactions here are compared with type I tRNAs having the short variable loop. The type II tRNAs are similar to the type I tRNAs in their tertiary base pairing interactions but differ from them generally by not having the tertiary base triples. The long variable loop, which is comprised of a helical stem and a loop at the end of it, emerges from the deep groove side of the dihydrouridine helix, and is tilted roughly 30° to the plane formed by the amino acid-pseudouridine and anticodon-dihydrouridine helices found in yeast tRNA^Phe. The fact that many of the type I tRNAs also lack the full compliment of base triples suggests that the tertiary base pairs may alone suffice to sustain the tRNA fold required for its biological function. The base triples and the variable loop appear to have little functional significance. The base type at position 9 is correlated with the number of base triples and G-C base pairs in the dihydrouridine stem.     

Mechanistic Studies on Metal-pyrophosphate Hydrolysis. Structure of Tetraammine(chloroaquo)cobait(III) dichloride ([CO(NH3)4ClH20]2+•-2Cl−), an Acid Hydrolysis Product of Co(NH3)4HP2O7 and Co(NH3)4PO4

Abstract

The octahedral complex tetraammine(chloroaquo)cobalt(III) dichloride is shown to be the HCl hydrolysis product of both P¹,^2-bidentate tetraammine(pyrophosphato)cobalt(III) [CO(NH₃)₄HP₂0₇ or CoPP] and bidentate tetraammine(phosphato)cobalt(III) [Co(NH₃)₄P0₄or CoP]. The complex crystallizes in the orthorhombic space group Pna2¹ with cell dimensions α=13.033(2)Å, b=6.710(1) Å, and c=10.318(2)Å; the crystal structure was refined to a final disagreement index of 0.033. The average of the four Co-N distances is 1.944±6Å. The Co-Cl distance is 2.257(2)Å and the Co-O(W) distance is 1.971(4)Å. Both protons of the coordinated water molecule are engaged in strong hydrogen bonds to the two nonbonded chloride counterions with 0(W)-C1 distances of 3.087(6)Å and 3.123(6)Å. Each nonbonded chloride is engaged in seven hydrogen bonding interactions resulting from the high ratio of hydrogen bond donors to acceptors in the CoP structure. Cobalt bisphosphate (CoP₂) is the final enzyme hydrolysis product when CoPP is used as substrate in the yeast inorganic pyrophosphatase reaction. The bridge oxygen atom is the site of initial CoPP cleavage both, for HCl catalyzed hydrolysis as well as for enzyme catalyzed hydrolysis.     

Two crystal forms of helix II of Xenopus laevis 5S rRNA with a cytosine bulge

The crystal structure of r(GCCACCCUG).r(CAGGGUCGGC), helix II of the Xenopus laevis 5S rRNA with a cytosine bulge (underlined), has been determined in two forms at 2.2 A (Form I, space group P4(2)2(1)2, a = b = 57.15 A and c = 43.54 A) and 1.7 A (Form II, space group P4(3)2(1)2, a = b = 32.78 A and c = 102.5 A). The helical regions of the nonamers are found in the standard A-RNA conformations and the two forms have an RMS deviation of 0.75 A. However, the cytosine bulge adopts two significantly different conformations with an RMS deviation of 3.9 A. In Form I, the cytosine bulge forms an intermolecular C+*G.C triple in the major groove of a symmetry-related duplex with intermolecular hydrogen bonds between N4C and O6G, and between protonated N3+C and N7G. In contrast, a minor groove C*G.C triple is formed in Form II with intermolecular hydrogen bonds between O2C and N2G, and between N3C and N3G with a water bridge. A partial major groove opening was observed in Form I structure at the bulge site. Two Ca2+ ions were found in Form I helix whereas there were none in Form II. The structural comparison of these two forms indicates that bulged residues can adopt a variety of conformations with little perturbation to the global helix structure. This suggests that bulged residues could function as flexible latches in bridging double helical motifs and facilitate the folding of large RNA molecules.