Preliminary crystallographic study of the alpha-D-galactose-specific lectin from jack fruit (Artocarpus integra) seeds

Crystals of the alpha-D-galactose-specific lectin from Jack fruit (Artocarpus integra) have been obtained from polyethylene glycol 400 solutions. The crystals are orthorhombic, space group P2(1)2(1)2 with a = 77.09 A, b = 123.3 A and c = 78.73 A (1 A = 0.1 nm) and have one 39,500 Mr tetramer per asymmetric unit. The crystals diffract to at least 2.8 A on precession photographs.     

Crystallization and preliminary crystallographic studies of an antitumour lectin from the edible mushroom Agrocybe aegerita

An antitumour lectin named AAL has been purified from the fruiting body of edible mushroom Agrocybe aegerita. In addition to having a distinct bioactivity, AAL shows strong inhibition effects on human and mouse tumour cells. It has been shown that AAL exerts its antitumour effects via apoptosis-induction. AAL and AAL-lactose complex have been crystallized and their diffraction data were collected with resolution of 2.6 A and 3.0 A, respectively. Both crystals belong to space group P 6122 with unit cell parameters a = 123.98 A, b = 123.98 A, c = 56.86 A, alpha= beta=90 degrees, gamma = 120 degrees and a = 123.69 A, b = 123.69 A, c = 56.64 A, alpha = beta = 90 degrees, gamma = 120 degrees, respectively.     

Crystallization and preliminary x-ray crystallographic analysis of galectin LEC-1 from Caenorhabditis elegans

Galectin LEC-1 isolated from the nematode Caenorhabditis elegans was the first galectin found in invertebrates and also the first tandem-repeat-type galectin identified, containing two homologous carbohydrate-binding sites. This galectin is localized most abundantly in the adult cuticle and possibly plays a role in the formation of epidermal layers. We succeeded in crystallizing LEC-1 composed of 279 amino acids with a calculated molecular weight of 31,809 Da under two independent sets of conditions as a result of extensive screening. The crystals grown under one set of conditions belong to the triclinic space group P1, with unit-cell parameters a = 48.44, b = 52.13, c = 64.24 A, alpha = 108.73, beta= 91.39, and gamma = 98.45 degrees and two protein molecules per unit cell. The crystals grown under the other set of conditions which included lactose belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.90, b = 47.01, c = 66.16 A, and beta= 113.30 degrees and one protein molecule per asymmetric unit.     

The active site of ribonuclease A from the crystallographic studies of ribonuclease-A-inhibitor complexes

Several members of the genus Methanosarcina were investigated by room-temperature and low-temperature difference spectroscopy for the presence of cytochromes. In combination with potentiometric titrations two membrane-bound b-cytochromes and one membrane-bound c-cytochrome could be detected in cells grown on methanol or trimethylamine. Very probably acetate-grown cells contained an additional cytochrome b. The midpoint potentials of the two b-type cytochromes were Em1 = -325 mV and Em2 = -183 mV, respectively. The additional b cytochrome formed during growth on acetate exhibited a midpoint potential of Em3 = -250 mV.     

Crystallization and preliminary crystallographic studies of the recombinant antitumour lectin from the edible mushroom Agrocybe aegerita

The antitumour lectin from Agrocybe aegerita, named AAL, shows strong inhibition effects on human and mouse tumour cells via apoptosis induction activity. Recombinant AAL (rAAL) has been expressed and purified. Both rAAL and rAAL-lactose complex have been crystallized and their X-ray diffraction data were collected to resolutions of 1.9 A and 1.6 A, respectively. Both crystals belong to space group P2(1) with unit cell parameters a = 53.20 A, b = 66.01 A, c = 57.86 A, beta = 109.38 and a = 53.38 A, b = 66.29 A, c = 58.02 A, beta = 109.03, respectively.     

Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes.

The crystal structures of rhizopuspepsin complexed with two oligopeptide inhibitors have been determined. CP-69,799, an azahomostatine dipeptide isostere, had previously been associated with a displacement of the C-terminal subdomain of endothiapepsin [Sali, A., Veerapandian, B., Cooper, J. B., Foundling, S. I., Hoover, D. J., & Blundell, T. L. (1989) EMBO J. 8, 2179-2188]. Here, we report the measurement of two data sets, one from crystals soaked in the inhibitor and the other from protein crystallized in the presence of excess inhibitor. In neither case is there any significant movement of the C-terminal subdomain of the rhizopuspepsin. The data suggest that the energy associated with any conformational change is small and is overcome by the crystal packing forces. The second inhibitor, a hydrated difluorostatone, was examined in a search for transition-state analogs that could cast further light on the mechanism of action [Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7009-7013]. The gem-diol provides a set of contact distances with the enzyme that mimic the interactions with the tetrahedral intermediate of the substrate during catalysis. These data provide support for the suggestion that the polarization of the keto group of the peptide substrate is enhanced by a hydrogen bond from the OD1 of Asp 35 (Suguna et al., 1987).     

Crystals of complexes mimicking protein biosynthesis are suitable for crystallographic studies

A complex of 70S ribosomes from Thermus thermophilus together with an average of 1.5-1.8 equivalents of PhetRNA(Phe) and a short mRNA chain, composed of 35 +/- 5 uridines, was crystallized under the conditions used for the growth of crystals of isolated ribosomes from the same source. Considering the reproducibility of their growth, their internal order and their shape, the crystals of the complex are superior to those of isolated ribosomes. In accord with previous three-dimensional reconstruction and modeling experiments, we conclude that the complex is less flexible and that an average population of complexes is more homogeneous than that of isolated 70S ribosomes. The crystals of the complex diffract to higher than 15 A resolution and can be irradiated with synchrotron X-ray beam at cryo-temperatures for days without noticeable decay. Since the crystals of the complex are apparently isomorphous with these of the isolated 70S ribosomes (P4(1)2(1)2; a = b = 526; c = 315 A), they should provide tool for phasing as well as for locating the mRNA and tRNA binding sites.     

Importance of nuclear localization for the apoptosis-induced activity of a fungal galectin AAL (Agrocybe aegerita lectin)

Agrocybe aegerita lectin (AAL) was identified previously in our group as a novel galectin from medicinal fungi Agrocybe aegerita, and has been shown to effectively induce cancer cell cycle arrest and apoptosis in vitro and tumor regression in vivo. Here, AAL was observed to translocate into the HeLa cell nucleus and induce cell apoptosis when it was predominantly in the nucleus. The N-terminus and C-terminus of AAL were required for nuclear localization. Site mutated proteins were generated based on AAL structure. Dimer interface mutant I25G, carbohydrate recognition domain (CRD) mutant R63H, and loop region mutant L33A could not enter the nucleus and lost the ability to induce apoptosis. CRD mutant H59Q and loop region mutant I144G maintained nuclear localization activity, and H59Q retained residual bioability but I144G had no activity, indicating that nuclear localization is important but not sufficient for AAL to become apoptotically active. Our findings provide a novel antitumor mechanism of fungal galectin.     

Synthetic, spectroscopic and X-ray crystallographic structural studies on copper(II) complexes of the aminoguanizone of pyruvic acid

Copper(II) complexes of general empirical formula, CuX(Hagpa) · nH₂O and Cu(agpa) · 2H₂O (H₂agpa = aminoguanizone of pyruvic acid, X = Cl⁻, Br⁻, , CH₃COO⁻, , n = 0, 1, 1.5, 2), have been synthesized and characterized by IR, EPR spectroscopy and X-ray crystallography. The IR spectra of the complexes showed the ONN coordination of the ligand to copper(II) ion. The crystal structures of H₂agpa · H₂O and complexes [Cu(Hagpa)Br] and [Cu₂(Hagpa)₂(H₂O)₂(SO₄)] · DMSO showed an invariable conformation and coordination mode for the uninegatively charged tridentate ligand and revealed the formation of linear polymers in which bromide or sulfate anions bridge the copper(II) ions. The EPR spectra for complexes CuX(Hagpa) · nH₂O are described by spin Hamiltonian for S = 1/2, without hyperfine structure. The g-tensor is symmetrical for Cu(agpa) · 2H₂O, has tri-axial anisotropy for sulfate complexes, and exhibits axial symmetry for the other compounds investigated.     

Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.     

Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS)

Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cyclical fashion within a trimeric unit. This arrangement has hindered the generation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the number of residues deleted and the penicillin substrate used. Crystal structures were determined for the DeltaR306 mutant complexed with iron(II) and 2-oxoglutarate (to 2.10 A) and the DeltaR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide (to 1.96 A). The latter may mimic a product complex, and supports proposals for a metal-bound CO(2) intermediate during catalysis.     

Capturing cyclic nucleotides in action: snapshots from crystallographic studies

Fifty years ago, cyclic AMP was discovered as a second messenger of hormone action, heralding the age of signal transduction. Many cellular processes were found to be regulated by cAMP and the related cyclic GMP. Cyclic nucleotides function by binding to and activating their effectors - protein kinase A, protein kinase G, cyclic-nucleotide-regulated ion channels and the guanine nucleotide-exchange factor Epac. Recent structural insights have now made it possible to propose a general structural mechanism for how cyclic nucleotides regulate these proteins.     

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the γ-phosphate of ATP to propionate during l-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate ≈ butyrate), nucleotides (ATP ≈ GTP > CTP ≈ TTP; dATP > dGTP > dCTP) and metal ions (Mg^2 + ≈ Mn^2 + > Co^2 +). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, α-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO₄ (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235–290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the γ-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer.     

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.