Crystallization and preliminary X-ray diffraction analysis of endoglucanase III from Trichoderma harzianum

Endoglucanases are enzymes that hydrolyze cellulose and are important components of the cellulolytic complex. In contrast to other members of the complex, they cleave internal beta-1,4-glycosidic bonds in the cellulose polymer, allowing cellulose to be used as an energy source. Since biomass is an important renewable source of energy, the structural and functional characterization of these enzymes is of interest. In this study, endoglucanase III from Trichoderma harzianum was produced in Pichia pastoris and purified. Crystals belonging to the orthorhombic space group P212121, with unit-cell parameters a = 47.54, b = 55.57, c = 157.3 angstrom, were obtained by the sitting-drop vapour-diffusion method and an X-ray diffraction data set was collected to 2.07 angstrom resolution.

Magnetic Tuning of All-Organic Binary Alloys between Two Stable Radicals

Mixtures of 2-(4,5,6,7-tetrafluorobenzimidazol-2-yl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl (F4BImNN) and 2-(benzi-midazol-2-yl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl (BImNN.) crystallize as solid solutions (alloys) across a wide range of binary compositions. (F4BImNN)(x)(BImNN)((1-x)) with x < 0.8 gives orthorhombic unit cells, while x >= 0.9 gives monoclinic unit cells. In all crystalline samples, the dominant intermolecular packing is controlled by one-dimensional (1D) hydrogen-bonded chains that lead to quasi-1D ferromagnetic behavior. Magnetic analysis over 0.4-300 K indicates ordering with strong 1D ferromagnetic exchange along the chains (J/k = 12-22 K). Interchain exchange is estimated to be 33- to 150-fold weaker, based on antiferromagnetic ordered phase formation below Neel temperatures in the 0.4-1.2 K range for the various compositions. The ordering temperatures of the orthorhombic samples increase linearly as (1 - x) increases from 0.25 to 1.00. The variation is attributed to increased interchain distance corresponding to decreased interchain exchange, when more F4BImNN is added into the orthorhombic lattice. The monoclinic samples are not part of the same trend, due to the different interchain arrangement associated with the phase change.

Base-Paired and Base-Stacked Structures of the Anti-HIV Drug Lamivudine: A Nucleoside DNA-Mimicry with Unprecedented Topology

We have prepared a DNA-mimicry of nucleosides in which the anti-HIV drug lamivudine (beta-L-2',3'-dideoxy-3'-thiacytidine, 3TC) self-assembles into a base-paired and helically base-stacked hexagonal structure. Face-to-face and face-to-tail stacked 3TC=3TC dimers base-paired through two hydrogen bonds between neutral cytosines by either N-H center dot center dot center dot O or N-H center dot center dot center dot N atoms give rise to a right-handed DNA-mimicry of lamivudine with an unusual highly symmetric hexagonal lattice and topology. In addition, a base-paired and base-stacked supramolecular architecture of lamivudine hemihydrochloride hemihydrate was also obtained as a result of our crystal screenings. This structure is formed through partially face-to-face stacked lamivudine pairs held together by protonated and neutral fragments. However, no helical stacking occurs in this structure in which lamivudine also adopts unusual conformations as the C1'-endo and C1'-exo sugar puckers and cytosine orientations intermediate between the anti and syn conformations. As a conclusion drawn from the nucleoside duplex, the hexagonal DNA-mimicry of lamivudine reveals that such double-stranded helices can be assembled without counterions and organic solvents but with higher crystallographic symmetry instead, because only water crystallizes together with lamivudine in this structure.

Crystal structure and spectroscopic properties of polymeric adducts of the tetra-mu-haloaspirinate-dicopper(II)

Eight new copper(II) complexes with halo-aspirinate anions have been synthesized: [Cu-2(Fasp)(4)(MeCN)(2)] center dot 2MeCN (1), [Cu-2(Clasp)(4)(MeCN)(2)]center dot 2MeCN (2), [Cu-2(Brasp)(4) (MeCn)(2)] center dot 2MeCn (3), {[Cu-2(Fasp)(4)(Pyrz)] center dot 2MeCN}(n) (4) {[Cu-2(Clasp)(4)(Pyrz)] center dot 2MeCN}(n) (5), [Cu-2(Brasp)(4)(Pyrz)](n) (6), [Cu-2(Clasp)(4)(4,4'-Bipy)](n) (7), and [Cu-2(Brasp)(4)(4,4'-Bipy)](n) (8) (Fasp: fluor-aspirinate; Clasp: chloro-aspirinate; Brasp: bromo-aspirinate; MeCN: acetonitrile; Pyrz: pyrazine; 4,4'-Bipy: 4,4'-bipyridine). The crystal structure of two 2 and 4 have been determined by X-ray diffraction methods. All compounds have been studied employing elemental analysis, IR, and UV-Visible spectroscopic techniques. The results have been compared with previous data reported for complexes with similar structures.

Crystal structure of dihydroorotate dehydrogenase from Leishmania major

Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases. (C) 2012 Elsevier Masson SAS. All rights reserved.

Synthesis, structures, redox and photophysical properties of benzodifuran-functionalised pyrene and anthracene fluorophores

Benzodifuran-functionalised pyrene and anthracene fluorophores 1 and 2 were obtained in reasonable yields. Their single crystal structures, electrochemical, optical absorption, and fluorescence characteristics have been described. They show strong luminescence with high quantum yields of 0.53 for 1 and 0.48 for 2.

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.

Syntheses and structures of molybdenum and tungsten complexes capable of epoxidaton and copper coordination polymers and dendrimers

We are interested in the syntheses of new complexes and in their characterization by single crystal X-ray diffraction techniques. Once we understand the structures, studies aimed at understanding uses of these complexes in the field of catalytic epoxidation using complexes soluble in water and syntheses of thin films (not assessed) were conducted. The syntheses, characterization and catalytic properties of a series of mononuclear, dinuclear and tetranuclear molybdenum and tungsten oxo complexes are described. The syntheses and structural characterization of two copper coordination polymers with 3,5-dihydroxylbenzoate ligand, and five paddlewheel shaped copper dendrimers coordinated with Fréchet-type dendrons are also detailed. The background of this dissertation is outlined in Chapter 1. Chapter 2 describes the syntheses, and characterization of two new mononuclear molybdenum(VI) and tungsten(VI) oxo complexes, MoO2Cl2(OPPh2CH2OH)2, and WO2Cl2(OPPh2CH2OH)2, bearing hydrophilic phosphine oxide ligand. The catalytic properties of these complexes for the epoxidation of cis-cyclooctene were also studied. Two new dinuclear molybdenum(VI) and tungsten(VI) oxo complexes Mo2O4Cl2[(HOCH2)PhPOO]2, and (CH3O)2(O)W(μ-O)(μ-O2PPh2)2W(O)(CH3O)2, bearing organophosphinate ligand are described in Chapter 3 and 4. Chapter 4 and 5 describes the syntheses and characterization of tetranuclear molybdenum(V) oxo complexes bearing various organophosphinate ligands. The catalytic abilities of these complexes for the epoxidation of cis-cyclooctene in the presence of hydrogen peroxide as oxidant were explored as well. Various spectroscopic methods, such as IR, UV-vis, and NMR are used to characterize the nature of these complexes. Crystal structures of compounds MoO2Cl2(OPPh2CH2OH)2, WO2Cl2(OPPh2CH2OH)2, Mo2O4Cl2[(HOCH2)PhPOO]2, (CH3O)2(O)W(μ-O)(μ-O2PPh2)2W(O)(CH3O)2, and Mo4(µ3-O)4(µ-O2PR2)4O4 (R=Ph, Me, ClCH2, o-C6H4(CH2)2) are also presented. The syntheses, and structural characterization of three copper(II) coordination polymers bearing 3,5-dihydroxybenzoate ligand are described in Chapter 6. Two copper(II) coordination polymers, [Cu2(3,5-dhb)2(pyridine)4]n, and [Cu2(3,5-dhb)4]n were afforded based on different amount of pyridine used in the reaction. The structures of these complexes are further built into 2D or 3D networks via inter or intra hydrogen bonds. The syntheses and structural characterization of the zinc(II) monomer, Zn(3,5-dhb)2(pyridine)2 is also described in this Chapter. Chapter 7 describes the syntheses, and characterization of five dendronized dicopper complexes bearing different generations of Fréchet-type dendrons. The structures of 3,5- bis(benzoyloxl)benzoic acid, 3,5-(PhCOO)2PhCOOH (G1), Cu2(3,5-dhb)4(THF)2, Cu2(G1)4(pyridine)2, and Cu2(G1)4(CH3OH)2 were characterized unambiguously by single X-ray diffraction. In addition, all compounds were characterized by FT-IR, UV-vis spectroscopy and elemental analyses.

Crystal chemistry and hydrogen bonding of rustumite Ca10(Si2O7)2(SiO4)(OH)2Cl2 with variable OH, Cl, F

Three samples of the skarn mineral rustumite Ca10(Si2O7)2(SiO4)(OH)2Cl2, space group C2/c, a ≈7.6, b ≈ 18.5, c ≈ 15.5 Å, β ≈ 104°, with variable OH, Cl, F content were investigated by electron microprobe, single-crystal X-ray structure refinements, and Raman spectroscopy. “Rust1LCl” is a low chlorine rustumite Ca10(Si2O7)2(SiO4)(OH1.88F0.12)(Cl1.28,OH0.72) from skarns associated with the Rize batholith near Ikizedere, Turkey. “Rust2F” is a F-bearing rustumite Ca10(Si2O7)2(SiO4)(OH1.13F0.87) (Cl1 96OH0.04) from xenoliths in ignimbrites of the Upper Chegem Caldera, Northern Caucasus, Russia. “Rust3LClF” represents a low-Cl, F-bearing rustumite Ca10(Si2O7)2(SiO4)0.87(H4O4)0.13(OH1.01F0.99) (Cl1.00 OH1.00) from altered merwinite skarns of the Birkhin massif, Baikal Lake area, Eastern Siberia, Russia. Rustumite from Birkhin massif is characterized by a significant hydrogarnet-like or fluorine substitution at the apices of the orthosilicate group, leading to specific atomic displacements. The crystal structures including hydrogen positions have been refined from single-crystal X-ray data to R1 = 0.0205 (Rust1_LCl), R1 = 0.0295 (Rust2_F), and R1 = 0.0243 (Rust3_LCl_F), respectively. Depletion in Cl and replacement by OH is associated with smaller unit-cell dimensions. The substitution of OH by F leads to shorter hydrogen bonds O-H⋯F instead of O-H⋯OH. Raman spectra for all samples have been measured and confirm slight strengthening of the hydrogen bonds with uptake of F.This study discusses the complex crystal chemistry of the skarn mineral rustumite and may provide a wider understanding of the chemical reactions related to contact metamorphism of limestones.

Crystal structure of the Anabaena sensory rhodopsin transducer.

We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed.

The Crystal Chemistry of Holtite

Holtite, approximately (Al,Ta,square)Al(6)(BO(3))(Si,Sb(3+),As(3+))(Sigma 3)O(12)(O,OH,square)(Sigma 3), is a member of the dumortierite group that has been found in pegmatite, or alluvial deposits derived from pegmatite, at three localities: Greenbushes, Western Australia; Voron'i Tundry, Kola Peninsula, Russia; and Szklary, Lower Silesia, Poland. Holtite can contain >30 wt.% Sb(2)O(3), As(2)O(3), Ta(2)O(5), Nb(2)O(5), and TiO(2) (taken together), but none of these constituents is dominant at a crystallographic site, which raises the question whether this mineral is distinct from dumortierite. The crystal structures of four samples from the three localities have been refined to R(1) = 0.02-0.05. The results show dominantly: Al, Ta, and vacancies at the Al(1) position; Al and vacancies at the Al(2), (3) and (4) sites; Si and vacancies at the Si positions; and Sb, As and vacancies at the Sb sites for both Sb-poor (holtite I) and Sb-rich (holtite II) specimens. Although charge-balance calculations based on our single-crystal structure refinements suggest that essentially no water is present, Fourier transform infrared spectra confirm that some OH is present in the three samples that could be measured. By analogy with dumortierite, the largest peak at 3505-3490 cm(-1) is identified with OH at the O(2) and O(7) positions. The single-crystal X-ray refinements and FTIR results suggest the following general formula for holtite: Al(7-[5x+y+z]/3)(Ta,Nb)(x)square([2x+y+z]/3)BSi(3-y)(Sb,As)(y)O(18-y-z)(OH)(z), where x is the total number of pentavalent cations, y is the total amount of Sb + As, and z <= y is the total amount of OH. Comparison with the electron microprobe compositions suggests the following approximate general formulae Al(5.83)(Ta,Nb)(0.50)square(0.67)BSi(2.50)(Sb,As)(0.50)O(17.00)(OH)(0.50) and Al(5.92)(Ta,Nb)(0.25)square(0.83)BSi(2.00)(Sb,As)(1.00) O(16.00)(OH)(1.00) for holtite I and holtite II respectively. However, the crystal structure refinements do not indicate a fundamental difference in cation ordering that might serve as a criterion for recognizing the two holtites as distinct species, and anion compositions are also not sufficiently different. Moreover, available analyses suggest the possibility of a continuum in the Si/(Sb + As) ratio between holtite I and dumortierite, and at least a partial continuum between holtite I and holtite II. We recommend that use of the terms holtite I and holtite II be discontinued.

Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2

Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 Å, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define β-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80°, 14° larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5° difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface.

Crystal structure of the ribosomal RNA domain essential for binding elongation factors

The structure of a 29-nucleotide RNA containing the sarcin/ricin loop (SRL) of rat 28 S rRNA has been determined at 2.1 Å resolution. Recognition of the SRL by elongation factors and by the ribotoxins, sarcin and ricin, requires a nearly universal dodecamer sequence that folds into a G-bulged cross-strand A stack and a GAGA tetraloop. The juxtaposition of these two motifs forms a distorted hairpin structure that allows direct recognition of bases in both grooves as well as recognition of nonhelical backbone geometry and two 5′-unstacked purines. Comparisons with other RNA crystal structures establish the cross-strand A stack and the GNRA tetraloop as defined and modular RNA structural elements. The conserved region at the top is connected to the base of the domain by a region presumed to be flexible because of the sparsity of stabilizing contacts. Although the conformation of the SRL RNA previously determined by NMR spectroscopy is similar to the structure determined by x-ray crystallography, significant differences are observed in the “flexible” region and to a lesser extent in the G-bulged cross-strand A stack.

Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors

Cytochrome P450 14α-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14α-methyl group from sterol precursors such as lanosterol, obtusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydrolanosterol. Inhibitors of CYP51 include triazole antifungal agents fluconazole and itraconazole, drugs used in treatment of topical and systemic mycoses. The 2.1- and 2.2-Å crystal structures reported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) are the first structures of an authentic P450 drug target. MTCYP51 exhibits the P450 fold with the exception of two striking differences—a bent I helix and an open conformation of BC loop—that define an active site-access channel running along the heme plane perpendicular to the direction observed for the substrate entry in P450BM3. Although a channel analogous to that in P450BM3 is evident also in MTCYP51, it is not open at the surface. The presence of two different channels, with one being open to the surface, suggests the possibility of conformationally regulated substrate-in/product-out openings in CYP51. Mapping mutations identified in Candida albicans azole-resistant isolates indicates that azole resistance in fungi develops in protein regions involved in orchestrating passage of CYP51 through different conformational stages along the catalytic cycle rather than in residues directly contacting fluconazole. These new structures provide a basis for rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.

Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors.

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.