Structure of the crystalline complex between ribonuclease A and D(pA)4.

Crystals of a complex formed between ribonuclease A and d(pA)4 were grown and their structure determined by a combination of multiple isomorphous replacement (MIR) and molecular replacement techniques. The known structure of ribonuclease A in the correct orientation in the unit cell yielded a conventional crystallographic R factor of 0.32 at 2.8 A resolution when refined as a rigid body. Difference Fourier syntheses permitted determination of the disposition of the DNA in the unit cell. Refinement of both protein and DNA by constrained-restrained least squares procedures resulted in an R factor of 0.22 at 2.5 A resolution. The structure of the crystalline complex is comprised of four ordered oligomers of d(pA)4 associated with each molecule of RNAse. If the sites of interaction between protein and d(pA)4 fragments are mapped on the surface of the protein, they describe an essentially continuous path into and through the active site, across the surface of the enzyme and finally into the basic amino acid cluster on the opposite side of the protein.     

Single molecule studies of DNA-protamine interactions

Single molecule studies of protamine-DNA interactions have characterized the kinetics of protamine binding to DNA and the morphology of the toroidal subunits that comprise sperm chromatin. The results provided by these studies are reviewed, the advantage of using single molecule techniques is discussed, and the implications of the results to the structure, kinetics of toroid formation, and stability of the DNA-protamine complex are described. New measurements of DNA condensation forces induced by the binding of protamine to DNA are also presented. These forces induce a significant tension in constrained segments of DNA and may contribute to the reduction in volume and shaping of the maturing spermatid cell nucleus.     

Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF.

Crystals of glutathione-S-transferase (GST)-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X-ray crystallographic analysis revealed that crystals of the GST-fused protein belong to space group P6(1)22 or P6(5)22 with unit cell dimensions a = b = 140.4 A, c = 93.5 A and gamma = 120 degrees, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 A resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA-binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well-ordered large crystals were unsuccessful. A possible application of GST-fusion proteins for small target proteins or domains to obtain crystals suitable for X-ray structure determination is proposed.     

Nonintercalative binding of proflavin to Z-DNA: structure of a complex between d(5BrC-G-5BrC-G) and proflavin

The crystal structure of a disordered 1:1 complex between the tetradeoxyoligomer d(5BrC-G-5BrC-G) and proflavin has been determined and refined to an R factor of 26.9% for 474 reflections initially in space group P6(5) and to an R factor of 22.2% for 475 reflections in space group P2(1), both at 2-A resolution with Fobsd greater than or equal to 4.0. The unit cell constants are a = b = 17.9 A, c = 44.5 A, and gamma = 120 degrees. The final models are essentially the same in the two space groups with greater disorder in space group P6(5). In space group P2(1), the asymmetric unit is a tetranucleotide duplex, two sandwiched proflavin molecules, and four "outside-bound" proflavins. The tetranucleotide duplex is in the Z conformation and is located at the origin of the unit cell with a pair of proflavins sandwiched between the tetranucleotides. Thus, the tetranucleotides and proflavin dimers stack alternatively forming a quasi-continuous helix with the helix axis coincident with the c axis. The structure analysis revealed the presence of outside-bound proflavins as well. It is interesting that one type of outside-bound proflavins occupies a similar environment as the cobalt hexaammines in their complex with the decadeoxyoligomer d(CGTACGTACG) [Brennan, R. G., Westhof, E., & Sundaralingam, M. (1986) J. Biomol. Struct. Dyn. 3, 649]. Crystals of the latter are isomorphous to the present complex. The outside-bound proflavins penetrate the deep minor groove, thereby closing it off, and provide a visualization of a quasi-internal mode of binding of proflavin to a nucleic acid.     

Lamellar bone: structure-function relations.

The term "bone" refers to a family of materials that have complex hierarchically organized structures. These structures are primarily adapted to the variety of mechanical functions that bone fulfills. Here we review the structure-mechanical relations of one bone structural type, lamellar bone. This is the most abundant type in many mammals, including humans. A lamellar unit is composed of five sublayers. Each sublayer is an array of aligned mineralized collagen fibrils. The orientations of these arrays differ in each sublayer with respect to both collagen fibril axes and crystal layers, such that a complex rotated plywood-like structure is formed. Specific functions for lamellar bone, as opposed to the other bone types, could not be identified. It is therefore proposed that the lamellar structure is multifunctional-the "concrete" of the bone family of materials. Experimentally measured mechanical properties of lamellar bone demonstrate a clear-cut anisotropy with respect to the axis direction of long bones. A comparison of the elastic and ultimate properties of parallel arrays of lamellar units formed in primary bone with cylindrically shaped osteonal structures in secondary formed bone shows that most of the intrinsic mechanical properties are built into the lamellar structure. The major advantages of osteonal bone are its fracture properties. Mathematical modeling of the elastic properties based on the lamellar structure and using a rule-of-mixtures approach can closely simulate the measured mechanical properties, providing greater insight into the structure-mechanical relations of lamellar bone.     

Structure refinement and diffuse streak scattering of silk (Bombyx mori).

Reexamination of the crystal structure of silk (Bombyx mori) was carried out by X-ray diffraction method. Four molecular chains are contained in the rectangular unit cell with parameters, a = 9.38 A, b = 9.49 A, and c (fiber axis) = 6.98 A, and the space group P2(1)-C(2)2. Silk assumes the statistical crystal structure, in which two antipolar-antiparallel sheet structures with different orientations statistically occupy a crystal site with the ratio 2:1. The molecular conformation is essentially the same pleated sheet structure as Marsh, Corey and Pauling proposed. However, the sheet structure formed by hydrogen bonds assumes the antipolar antiparallel structure different from that proposed by Marsh, Corey and Pauling, in which the methyl groups of alanine residues alternately point to both sides of the sheet structure along the hydrogen bonding direction. The crystalline region of silk is composed of stacking of two antipolar antiparallel sheet structures with different orientations.     

Structure, flexibility, and mechanism of the Bacillus stearothermophilus RecU Holliday junction resolvase

Here we report a high resolution structure of RecU-Holliday junction resolvase from Bacillus stearothermophilus. The functional unit of RecU is a homodimer that contains a "mushroom" like structure with a rigid cap and two highly flexible loops extending outwards. These loops appear to be highly flexible/dynamic, and presumably are directly involved in DNA binding and holding it for catalysis. Structural modifications of both the protein and DNA upon their interaction are essential for catalysis. An Mg2+ ion is present in each of the two active sites in this homodimeric enzyme, and two water molecules are coordinated with each Mg2+ ion. Our data are consistent with one of these water molecules acting as a nucleophile and the other as a general acid. The identities of the general base and general acid involved in catalysis and the Lewis acid that stabilizes the pentacovalent transition state phosphate ion are proposed. A model for the RecU-Holliday junction DNA complex is also proposed and discussed in the context of DNA binding and cleavage.     

Biostructural analysis of the metal-sensor domain of CnrX from <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34

In Cupriavidus metallidurans CH34, the proteins CnrX, CnrY, and CnrH regulate the expression of the cnrCBA operon that codes for a cation-efflux pump involved in cobalt and nickel resistance. The periplasmic part of CnrX can be defined as the metal sensor in the signal transduction complex composed of the membrane-bound anti-sigma factor CnrY and the extra-cytoplasmic function sigma factor CnrH. A soluble form of CnrX was overproduced and purified. This protein behaves as a dimer in solution as judged from gel filtration, sedimentation velocity experiments, and NMR. Native crystals diffracting to 2.3 A using synchrotron radiation were obtained using the hanging-drop vapor-diffusion method. They belong to the primitive monoclinic space group P2(1), with unit cell parameters a = 31.87, b = 74.80, c = 93.67 A, beta = 90.107 degrees. NMR data and secondary structure prediction suggest that this protein is essentially formed by helices.     

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).     

Determining the crystal structure of cellulose III(I) by modeling

Recently, a one-chain monoclinic unit cell for cellulose III(I) having P2(1) symmetry and a single glucose in the asymmetric unit was proposed, based on high-resolution diffraction patterns. The new work challenged a two-chain structure that was published 25 years earlier, although it did not provide new three-dimensional coordinates. Our goals were to solve the structure by modeling, find whether modeling would reject the previously determined two-chain unit cell, and compare the model with the anticipated experimental structure. Combinations of three rotamers of the O-2, O-3, and O-6 hydroxyl groups produced 27 'up' and 27 'down' starting structures. Clusters ('minicrystals') of 13 cellotetraose chains terminated by methyl groups for each of the 54 starting structures were optimized with MM3(96). Hydroxyl groups on 16 of these 54 structures reoriented to give very similar hydrogen-bonding schemes in the interiors, along with the lowest energies. Hydrogen bonds included the usual intramolecular O-3H...O-5' linkage, with O-6' also accepting from O-3H. Interchain hydrogen bonds form an infinite, cooperative O-6H...O-2H...O-6 network. Direct comparison of total minicrystal energies for the one- and two-chain unit cell was inappropriate because the two-chain cell's alternate chains are shifted 0.9 A along the z-axis. To get comparable energy values, models were built with both cellotetraose and cellohexaose chains. The differences in their energies represent the energies for the central layers of cellobiose units. The one-chain cell models had much lower energy. The eight best 'up' one-chain models agree reasonably well with the structure newly determined by experiment.     

Cloning and analysis of the gene (rpoDA) for the principal sigma factor of Pseudomonas aeruginosa

A gene (rpoDA) of Pseudomonas aeruginosa whose gene product has a homologous function and structure with the principal sigma factor of Escherichia coli was cloned and sequenced. The DNA region corresponding to one of the two hybridization signals found in P. aeruginosa DNA with a synthetic oligonucleotide probe (rpoD probe) was shown to be able to complement a temperature sensitive mutation of Escherichia coli rpoD gene. The amino acid sequence deduced from the nucleotide sequence of rpoDA showed an extensive homology with that of the principal sigma factor of E. coli throughout the entire region, which indicates that the two gene products have an essentially identical domain structure. A common basic structure observed among principal sigma factors of different eubacterial strains was proposed. RpoDA protein was identified in the extract of the cell carrying a plasmid clone with the rpoDA gene insert by Western blot analysis.     

Crystal structure of chartreusin derivative A132

The crystal structure of chartreusin derivative A132 (benzilidene chartreusin) has been determined by single-crystal X-ray diffraction. The space group is C2 with unit cell dimensions, a=18.482(4), b=8.749(3), c=43.906(2) A, beta=94.87(2) degrees, and the structure was refined to R-factors of 0.2365 (6585 all unique reflections) and 0.087 (2914 reflections with F(o)>4 sigma(F(o))) by a full-matrix least-squares method. There are two molecules in an asymmetric unit. Both molecules have similar structures, which are favorable to bind with DNA in the minor groove. A modeling study of the A132-DNA complex based on the X-ray structures suggests that the sugar moiety of A132 may play an important role in recognizing the sequence of DNA base pairs.     

Crystal structures of cyclomaltohexaose (alpha-cyclodextrin) complexes with p-bromophenol and m-bromophenol.

Crystal structures of cyclomaltohexose (alpha-cyclodextrin) complexes with p-bromophenol and m-bromophenol have been determined by single-crystal X-ray diffraction. The space group of the alpha-cyclodextrin-p-bromophenol complex is P2(1)2(1)2(1) with unit cell dimensions of a = 15.318(3), b = 24.733(3), c = 13.457(2) A, and that of the alpha-cyclodextrin-m-bromophenol complex is P2(1)2(1)2 with unit cell dimensions of a = 25.858(7), b = 27.263(8), c = 8.145(3) A. In crystals, the alpha-cyclodextrin-p-bromophenol complex and the alpha-cyclodextrin-m-bromophenol complex form a layer-type and a channel-type molecular packing structure, respectively. The intermolecular hydrogen-bond interactions of the hydroxyl groups of bromophenols are closely related to the molecular packing structure.     

The cytosol origin of macromolecules extruded by cultured chick embryo fibroblast cells

The DNA and protein extruded by chick embryo fibroblast cells has been analysed by chromatography. A high proportion of the DNA is in the form of a protein-complex of size around 5 X 10(5) dalton. The patterns of the DNA and protein extruded into the supernatant are closely similar in many respects to those found in the cell cytosol. It is concluded that the macromolecular material extruded by cells in culture is of cytosol origin: a possible function in terms of "information" carriage is proposed.     

A dual chromatin organization in the sperm of the bivalve mollusc Spisula solidissima

Most of the DNA in the sperm of the bivalve mollusc. Spisula solidissima, is found to be associated with a specific high-molecular-mass, protamine-like component, sharing features common both to protamines and to histones. We have found that this component coexists, in the mature sperm nucleus, with a complete set of histones, including an H1-like histone. Such histones account for approximately 20% of the whole protein content in the sperm chromatin, the overall protein/DNA ratio (w/w) being 0.87. These data, together with micrococcal nuclease digestions in combination with salt fractionation, have allowed us to propose a structural model for this chromatin in which short nucleosomal domains are interspersed in a highly saturated protamine-DNA complex.     

Visualization of drug-nucleic acid interactions at atomic resolution. IX. Structures of two N,N-dimethylproflavine: 5-iodocytidylyl (3'-5') guanosine crystalline complexes

This paper describes two complexes containing N,N-dimethylproflavine and the dinucleoside monophosphate, 5-iodocytidylyl (3'-5') guanosine (iodoCpG). The first complex is triclinic, space group P1, with unit cell dimensions a = 11.78 A, b = 14.55 A, c = 15.50 A, alpha = 89.2 degrees, beta = 86.2 degrees, gamma = 96.4 degrees. The second complex is monoclinic, space group P21, with a = 14.20 A. b = 19.00 A, c = 20.73 A, beta = 103.6 degrees. Both structures have been solved to atomic resolution and refined by Fourier and least squares methods. The first structure has been refined anisotropically to a residual of 0.09 on 5,025 observed reflections using block diagonal least squares, while the second structure has been refined anisotropically to a residual of 0.13 on 2,888 reflections with full matrix least squares. The asymmetric unit in both structures contains two dimethylproflavine molecules and two iodoCpG molecules; the first structure has 16 water molecules (a total of 134 non-hydrogen atoms), while the second structure has 18 water molecules (a total of 136 non-hydrogen atoms). Both structures demonstrate intercalation of dimethylproflavine between base-paired iodoCpG dimers. In addition, dimethylproflavine molecules stack on either side of the intercalated duplex, being related by a unit cell translation along b and a axes, respectively. The basic structural feature of the sugar-phosphate chains accompanying dimethylproflavine intercalation in both structures is the mixed sugar puckering pattern: C3' endo (3'-5') C2' endo. This same structural information is again demonstrated in the accompanying paper, which describes a complex containing dimethylproflavine with deoxyribo-CpG. Similar information has already appeared for other "simple" intercalators such as ethidium, acridine orange, ellipticine, 9-aminoacridine, N-methyl-tetramethylphenanthrolinium and terpyridine platinum. "Complex" intercalators, however, such as proflavine and daunomycin, have given different structural information in model studies. We discuss the possible reasons for these differences in this paper and in the accompanying paper.     

Crystallization and preliminary X-ray analysis of receptor-binding protein P2 of bacteriophage PRD1

Bacteriophage PRD1 has remarkable structural similarities to adenovirus, but is unusual in containing a membrane beneath its icosahedral capsid. Its monomeric receptor-binding protein, P2, is part of a complex at each capsid vertex and so is the functional equivalent of adenovirus fiber. P2 has been crystallized by the "hanging-drop" method of vapor diffusion and two different crystal forms were obtained. Macroseeding, used to increase the size of the initial small needles, gave rod-shaped crystals. These grew to a size of 0.08 x 0.08 x 0.50 mm(3) and diffracted to 2.6 A resolution. They have the orthorhombic space group P222(1), with unit cell dimensions a = 137.8 A, b = 46.5 A, c = 136.4 A. A few single crystals of a second form were grown without seeding under slightly different conditions. A parallelepiped crystal (0.10 x 0.10 x 0.35 mm(3)), with space group C222(1) and unit cell dimensions a = 182.3 A, b = 204.8 A, c = 133.3 A, diffracted to 3.5 A resolution. A rotation function for the second form revealed that four monomers of P2 are related by a noncrystallographic twofold axis. The structure of P2 will reveal how this arrangement relates to the trimeric adenovirus fiber.