Julia M. Richardson

Molecular architecture of the Mos1 paired-end complex: the structural basis of DNA transposition in a eukaryote

A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, with each transposon end recognized by the DNA binding region of one transposase monomer and by the active site of the other monomer. Two additional DNA duplexes in the crystal indicate likely binding sites for flanking DNA. Biochemical data provide support for a model of the target capture complex and identify Arg186 to be critical for target binding. Mixing experiments indicate that a transposase dimer initiates first-strand cleavage and suggest a pathway for PEC formation.

The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei.

Trypanosoma brucei, in common with the other African trypanosomes, exhibits unusual cell-surface molecular architecture. The bloodstream form of the parasite is coated with a continuous layer of approximately five million variant surface glycoprotein (VSG) dimers that provide the parasite with a macromolecular diffusion barrier to guard against lysis by the alternative complement pathway. The procyclic form of the parasite has a more diffuse cell-surface coat made up of approximately 2.5 million copies of procyclic acidic repetitive protein (PARP). Within the VSG and PARP coats exist lower-abundance surface glycoproteins such as receptors and nutrient transporters. Both the VSG molecules and the PARP molecules are attached to the membrane via glycosylphosphatidylinositol (GPI) membrane anchors and the VSGs and one form of PARP are N-glycosylated. In this article, the structures of the N-glycans and the GPI anchors of T. brucei VSGs and PARPs are reviewed and simple models of the surfaces of bloodstream and procyclic trypomastigotes are presented.

Mechanism of Mos1 transposition: insights from structural analysis.

We present the crystal structure of the catalytic domain of Mos1 transposase, a member of the Tc1/mariner family of transposases. The structure comprises an RNase H‐like core, bringing together an aspartic acid triad to form the active site, capped by N‐ and C‐terminal α‐helices. We have solved structures with either one Mg2+ or two Mn2+ ions in the active site, consistent with a two‐metal mechanism for catalysis. The lack of hairpin‐stabilizing structural motifs is consistent with the absence of a hairpin intermediate in Mos1 excision. We have built a model for the DNA‐binding domain of Mos1 transposase, based on the structure of the bipartite DNA‐binding domain of Tc3 transposase. Combining this with the crystal structure of the catalytic domain provides a model for the paired‐end complex formed between a dimer of Mos1 transposase and inverted repeat DNA. The implications for the mechanisms of first and second strand cleavage are discussed.

Trypanosoma brucei Glycoproteins Contain Novel Giant Poly-N-acetyllactosamine Carbohydrate Chains

The flagellar pocket of the bloodstream form of the African sleeping sickness parasite Trypanosoma brucei contains material that binds the β-d-galactose-specific lectin ricin (Brickman, M. J., and Balber, A. E. (1990) J. Protozool. 37, 219–224). Glycoproteins were solubilized from bloodstream form T. brucei cells in 8 m urea and 3% SDS and purified by ricin affinity chromatography. Essentially all binding of ricin to these glycoproteins was abrogated by treatment with peptide N-glycosidase, showing that the ricin ligands are attached to glycoproteins via N-glycosidic linkages to asparagine residues. Glycans released by peptide N-glycosidase were resolved by Bio-Gel P-4 gel filtration into two fractions: a low molecular mass mannose-rich fraction and a high molecular mass galactose and N-acetylglucosamine-rich fraction. The latter fraction was further separated by high pH anion exchange chromatography and analyzed by gas chromatography mass spectrometry, one- and two-dimensional NMR, electrospray mass spectrometry, and methylation linkage analysis. The high molecular mass ricin-binding N-glycans are based on a conventional Manα1–3(Manα1–6)Manβ1–4-GlcNAcβ1–4GlcNAc core structure and contain poly-N-acetyllactosamine chains. A significant proportion of these structures are extremely large and of unusual structure. They contain an average of 54 N-acetyllactosamine (Galβ1–4GlcNAc) repeats per glycan, linked mostly by -4GlcNAcβ1–6Galβ1-interrepeat linkages, with an average of one -4GlcNAcβ1–3(-4GlcNAcβ1–6)Galβ1- branch point in every six repeats. These structures, which also bind tomato lectin, are twice the size reported for the largest mammalian poly-N-acetyllactosamine N-linked glycans and also differ in their preponderance of -4GlcNAcβ1–6Galβ1- over -4GlcNacβ1–3Galβ1- interrepeat linkages. Molecular modeling suggests that -4GlcNAcβ1–6Galβ1- interrepeat linkages produce relatively compact structures that may give these giant N-linked glycans unique physicochemical properties. Fluorescence microscopy using fluorescein isothiocyanatericin indicates that ricin ligands are located mainly in the flagellar pocket and in the endosomal/lysosomal system of the trypanosome.

ASSESSMENT OF A METHOD FOR THE MEASUREMENT OF LONG-RANGE HETERONUCLEAR COUPLING-CONSTANTS

Abstract Recently ( J. Magn. Reson. 85 , 111–131, 1989) we described a method for the convenient measurement of long-range heteronuclear coupling constants using a combination of a proton-detected two-dimensional shift-correlation spectrum and a simple data-fitting procedure. This method enables accurate values of the long-range couplings to be extracted from the highly distorted cross peaks in the two-dimensional spectrum. In this paper the reliability of the method is investigated to establish likely error limits on the values of the couplings. The effects of strong coupling, isotopic labeling, phase variations, thermal noise, and t 1 noise are all considered. It is concluded that the procedure is robust with respect to deviations of the spectra from the ideal and, for typical experimental data, yields values of coupling constants which have errors of less than 10%.

Chemical structure of Trichomonas vaginalis surface lipoglycan: a role for short galactose (β1-4/3) N-acetylglucosamine repeats in host cell interaction

Background: Trichomonas vaginalis lipoglycan (TvLG) mediates interactions between the parasite and human host. Results: TvLG is composed of a polyrhamnose backbone with branches of poly-N-acetyllactosamine that are involved in attachment to host epithelium. Conclusion: TvLG has a unique structure among solved parasite glycans. Significance: This work provides a template to analyze TvLG from T. vaginalis with different binding properties. The extracellular parasite Trichomonas vaginalis contains a surface glycoconjugate that appears to mediate parasite-host cell interaction via binding to human galectin-1. This glycoconjugate also elicits cytokine production from human vaginal epithelial cells, implicating its role in modulation of host immune responses. We have analyzed the structure of this glycoconjugate, previously described to contain the sugars rhamnose (Rha), N-acetylglucosamine (GlcNAc), galactose (Gal), xylose (Xyl), N-acetylgalactosamine (GalNAc), and glucose (Glc), using gas chromatograph mass spectrometry (GC-MS), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), electrospray MS/MS, and nuclear magnetic resonance (NMR), combined with chemical and enzymatic digestions. Our data reveal a complex structure, named T. vaginalis lipoglycan (TvLG), that differs markedly from Leishmania lipophosphoglycan and Entamoeba lipopeptidophosphoglycan and is devoid of phosphosaccharide repeats. TvLG is composed of an α1–3 linked polyrhamnose core, where Rha residues are substituted at the 2-position with either β-Xyl or chains of, on average, five N-acetyllactosamine (-3Galβ1–4GlcNAcβ1-) (LacNAc) units and occasionally lacto-N-biose (-3Galβ1-3GlcNAcβ1-) (LNB). These chains are themselves periodically substituted at the Gal residues with Xyl-Rha. These structural analyses led us to test the role of the poly-LacNAc/LNB chains in parasite binding to host cells. We found that reduction of poly-LacNAc/LNB chains decreased the ability of TvLG to compete parasite binding to host cells. In summary, our data provide a new model for the structure of TvLG, composed of a polyrhamnose backbone with branches of Xyl and poly-LacNAc/LNB. Furthermore, the poly-LacNAc side chains are shown to be involved in parasite-host cell interaction.

Solution conformations of early intermediates in Mos1 transposition.

DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly.

Structures of Leishmania major orthologues of macrophage migration inhibitory factor

Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, expresses two closely related MIF-like proteins. To ascertain the roles and potential differences of these two Leishmania proteins, recombinant L. major MIF1 and MIF2 have been produced and the structures resolved by X-ray crystallography. Each has a trimeric ring architecture similar to mammalian MIF, but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response, thereby promoting parasite survival, but suggest the LmjMIFs have potentially different biological roles. Analysis of the Leishmania braziliensis genome showed that this species lacks both MIF genes. Thus MIF is not a virulence factor in all species of Leishmania.

Biochemical and immunological characterization of Toxoplasma gondii macrophage migration inhibitory factor.

Background: Toxoplasma gondii possesses proteins that can modulate host immune responses. TgMIF is a homologue of mammalian macrophage migration inhibitory factor. Results: TgMIF possesses tautomerase but not oxidoreductase activity. It can stimulate ERK MAPK and IL-8 production. Conclusion: TgMIF is capable of modulating host molecules. Significance: The study identifies immunomodulatory activity of TgMIF consistent with a role for sustaining parasite survival. Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 μmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

Expression, purification and preliminary crystallographic studies of a single-point mutant of Mos1 mariner transposase

A soluble single-point mutant of full-length Mos1 mariner transposase (MW = 40.7 kDa) has been overexpressed in Escherichia coli, purified to 95% homogeneity and crystallized. This provides the first example of the crystallization of a eukaryotic transposase. The native crystals diffract to 2.5 A resolution and show tetragonal symmetry, with unit-cell parameters a = b = 44.5, c = 205.6 A. Multiple-wavelength anomalous data from a selenomethionyl form of the protein and data from a heavy-atom derivative have been collected.