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1.  Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling 
The Journal of General Virology  2013;94(Pt 12):2777-2789.
Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.
doi:10.1099/vir.0.057729-0
PMCID: PMC3836500  PMID: 24088344
2.  Resonance assignments for latherin, a natural surfactant protein from horse sweat 
Latherin is an intrinsically surfactant protein of ~23 kDa found in the sweat and saliva of horses. Its function is probably to enhance the translocation of sweat water from the skin to the surface of the pelt for evaporative cooling. Its role in saliva may be to enhance the wetting, softening and maceration of the dry, fibrous food for which equines are adapted. Latherin is unusual in its relatively high content of aliphatic amino acids (~25 % leucines) that might contribute to its surfactant properties. Latherin is related to the palate, lung, and nasal epithelium carcinoma-associated proteins (PLUNCs) of mammals, at least one of which is now known to exhibit similar surfactant activity to latherin. No structures of any PLUNC protein are currently available. 15N,13C-labelled recombinant latherin was produced in Escherichia coli, and essentially all of the resonances were assigned despite the signal overlap due to the preponderance of leucines. The most notable exceptions include a number of residues located in an apparently dynamic loop region between residues 145 and 154. The assignments have been deposited with BMRB accession number 19067.
doi:10.1007/s12104-013-9485-3
PMCID: PMC3955484  PMID: 23708874
Latherin; Surfactant protein; Horse; Sweat; Saliva; Allergen; NMR
3.  Resonance assignment of As-p18, a fatty acid binding protein secreted by developing larvae of the parasitic nematode Ascaris suum 
As-p18 is produced and secreted by larvae of the parasitic nematode Ascaris suum as they develop within their eggs. The protein is a member of the fatty acid binding protein (FABP) family found in a wide range of eukaryotes, but is distinctive in that it is secreted from the synthesizing cell and has predicted additional structural features not previously seen in other FABPs. As-p18 and similar proteins found only in nematodes have therefore been designated ‘nemFABPs’. Sequence-specific 1H, 13C and 15N resonance assignments were established for the 155 amino acid recombinant protein (18.3 kDa) in complex with oleic acid, using a series of three-dimensional triple-resonance heteronuclear NMR experiments. The secondary structure of As-p18 is predicted to be very similar to other FABPs, but the protein has extended loops that have not been observed in other FABPs whose structures have so far been solved.
doi:10.1007/s12104-012-9447-1
PMCID: PMC3955487  PMID: 23225165
As-p18; Fatty acid binding protein; nemFABP; Nematode; Parasite; Ascaris suum
4.  Plant UVR8 Photoreceptor Senses UV-B by Tryptophan-Mediated Disruption of Cross-Dimer Salt Bridges 
Science (New York, N.Y.)  2012;335(6075):1492-1496.
The recently identified plant photoreceptor UVR8 triggers regulatory changes in gene expression in response to ultraviolet-B (UV-B) light via an unknown mechanism. Here, crystallographic and solution structures of the UVR8 homodimer, together with mutagenesis and far-UV circular dichroism spectroscopy, reveal its mechanisms for UV-B perception and signal transduction. β-propeller subunits form a remarkable, tryptophan-dominated, dimer interface stitched together by a complex salt-bridge network. Salt-bridging arginines flank the excitonically coupled cross-dimer tryptophan “pyramid” responsible for UV-B sensing. Photoreception reversibly disrupts salt bridges, triggering dimer dissociation and signal initiation. Mutation of a single tryptophan to phenylalanine re-tunes the photoreceptor to detect UV-C wavelengths. Our analyses establish how UVR8 functions as a photoreceptor without a prosthetic chromophore to promote plant development and survival in sunlight.
doi:10.1126/science.1218091
PMCID: PMC3505452  PMID: 22323738
5.  1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus 
The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.
doi:10.1007/s12104-012-9444-4
PMCID: PMC3955486  PMID: 23179061
Parasitic nematode; Necator americanus; Fatty-acid and retinol-binding protein; Na-FAR-1; NMR
6.  Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline 
As-p18, an unusual fatty-acid-binding protein from a parasitic nematode, was expressed in bacteria, purified and crystallized. The use of a microfocus beamline was essential for data collection.
As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of ‘microdomains’. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8 µm.
doi:10.1107/S1744309112026553
PMCID: PMC3412778  PMID: 22869127
fatty-acid-binding proteins; parasitic nematodes; Ascaris suum; microfocus beamlines
7.  Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus  
Na-FAR-1, a fatty acid- and retinol-binding protein, was expressed in bacteria, purified and crystallized. Crystals grew in two different morphologies under the same conditions.
Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning.
doi:10.1107/S1744309112023597
PMCID: PMC3388935  PMID: 22750878
fatty acid- and retinol-binding proteins; parasitic nematodes; Necator americanus; Na-FAR-1
9.  The role of conserved residues of chagasin in the inhibition of cysteine peptidases 
Febs Letters  2008;582(4-3):485-490.
We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10–100-fold increases in the Ki for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.
doi:10.1016/j.febslet.2008.01.008
PMCID: PMC2607524  PMID: 18201565
Z-Phe-Arg-MCA, carbobenzoxy-phenylalanyl-arginyl-7-amido-4-methylcoumarin; PBS, phosphate buffered saline; cruzain, recombinant cruzipain truncated at the C-terminal extension; DTT, dithiothreitol; EDTA, ethylenidiaminetetracetic acid disodium salt 2-hydrate; E-64, l-trans-epoxysuccinylleucylamido-(4-guanidino) butane; IPTG, isopropyl-β-d-thiogalactopyranoside; Chagasin; Cysteine peptidase; Inhibitor; Mutant; Trypanosoma
10.  THE STRUCTURE OF LEISHMANIA MEXICANA ICP PROVIDES EVIDENCE FOR CONVERGENT EVOLUTION OF CYSTEINE PEPTIDASE INHIBITORS* 
The Journal of biological chemistry  2005;281(9):5821-5828.
Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens, but are absent from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes of CP inhibitor. Recent data suggest that L. mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicanaby NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria. The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile. Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism.
doi:10.1074/jbc.M510868200
PMCID: PMC1473161  PMID: 16407198

Results 1-10 (10)