The VACV A46 protein constructs and purification of A46(1–83).

<p>A, Structural alignment of VACV Bcl-2-like immunomodulators. The structural alignment was generated using the T-coffee online algorithm [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref022" target="_blank">22</a>] with additional manual correction. Protein Data Bank (PDB) codes are 4LQK, 4M0S (A46, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref009" target="_blank">9</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref023" target="_blank">23</a>]), 2VVW (A52, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref010" target="_blank">10</a>]), 2VVY (B14, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref010" target="_blank">10</a>]), 4D5S (A49, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref011" target="_blank">11</a>]), 3JRV (K7, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref014" target="_blank">14</a>]), 2I39 (N1, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref012" target="_blank">12</a>]), 2VTY, 4D2L (F1, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref015" target="_blank">15</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref016" target="_blank">16</a>]). Bullets indicate the residues forming the hydrophobic core of the Bcl-2-like domains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref007" target="_blank">7</a>]. Portions of proteins seen in three dimensional protein structures are highlighted in grey, helices in red and β-strands in cyan. B, Schematic representation of expression constructs for the A46 N-terminal domain. HIS, hexahistidine tag. TRX, thioredoxin solubilisation tag (green). TEV, TEV protease cleavage site. A46 variants (light blue). C, SDS-PAGE analysis of the purification of A46(1–83). Lane 1, crude cell extract (T); lane 2, total soluble protein in crude cell extract loaded on to Ni-NTA beads (S); lane 3, flow-through from Ni-NTA beads (FT); lane 4, eluate from Ni-NTA beads with imidazole (E); lane 5, eluate after incubation with TEV protease (E+TEV); lane 6, 15 μg of final product from concentrated pooled fraction after SEC. The gel contained 15% acrylamide; proteins were visualized with Coomassie Brilliant Blue R250. Apparent molecular sizes (in kDa) are indicated on the left.</p

Inhibitory effect of full-length and truncated constructs of A46 on the IL-1β stimulated NK-κB transcription.

<p>HEK293T cells were transfected with the indicated amounts of plasmids. Plasmid amounts were adjusted so that approximately the same amounts of each A46 variant were expressed. After 40 h of incubation, cells were stimulated with 0.32 ng/ml of IL-1β and incubated for a further 6 h. NF-κB reporter gene activity was then measured. Data are expressed as the relative stimulation from a representative experiment from a minimum of three separate experiments, each performed in triplicate. Error bars represent the standard deviation from the mean. EV, empty vector.</p

Overall structure of the N-terminal domain of A46.

<p>A, Two views of the structure of A46(1–83). The dimer comprises subunit A (7 β-strands) and subunit B (6 β-strands) with the subunits coloured as rainbows from the N- to the C-termini. Myristic acid co-crystallized as a ligand inside of the cavity and is depicted as sticks; carbon atoms are blue and oxygen ones are red. B, Superimposition of subunit B on subunit A of A46(1–83) dimer. Subunit A (in blue) and subunit B (in red) were superimposed in PyMOL. MYR, myristic acid. The amino acids most differing within subunits are indicated. Panel B is related to panel A by a counter-clockwise rotation of 30°. Drawings were made using PyMOL [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref029" target="_blank">29</a>].</p

Analysis of binding of A46 variants to TIR domains of MyD88, MAL and TRAM (K_D, μM).

<p>Analysis of binding of A46 variants to TIR domains of MyD88, MAL and TRAM (K_D, μM).</p

Identification of the fatty acids co-purified with A46 constructs.

<p>A, Stereo view of myristic acid accommodated in a hydrophobic cavity of A46(1–83) dimer. <i>2F</i>_<i>0</i><i>-F</i>_<i>c</i> electron density map of myristic acid contoured at 1σ. Subunit A is blue, subunit B is red. Myristic acid is depicted as sticks, hydrophobic residues are coloured in the colour of respective subunit. Drawings were made using PyMOL [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#ppat.1006079.ref029" target="_blank">29</a>]. B, C, D Total fatty acid content of fatty acids present in the extract from A46(1–83), A46(1–229) or A46(87–229), respectively. Fatty acids released by base and converted to methyl esters and analysed by GC-MS (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006079#sec012" target="_blank">material and methods</a> for details). The figure shows the TIC chromatogram 31–38 min in which the identified fatty acid methyl ester species are marked. E, Typical content of fatty acid extract from <i>E</i>.<i>coli</i> DE3 cells grown in LB medium at 25°C.</p

SAXS analysis of the N-terminal domain of A46(1–83) and full-length A46(1–240).

<p>A, SAXS analysis of the N-terminal domain of A46 (1–83). Experimental scattering profile is shown with black dots, theoretical scattering curve of A46(1–83) tetramer formed by a dimer in the asymmetric unit and its symmetry mate is in red and the one from the asymmetric unit dimer in green. The graph representing P(r) function with indicated D_max is in the center and Guinier plot on the right. B, SAXS analysis of the full-length A46(1–240). Experimental scattering profile is shown with black dots, calculated scattering curves from SAXS models are presented in blue (<i>ab initio</i> model calculated by DAMMIF) and in green (rigid body modeling by CORAL). The graph representing P(r) function with indicated D_max is in the center and Guinier plot on the right. C, Superimposition of the <i>ab initio</i> model envelope (grey surface) with the rigid-body modeling of the N- and C-terminal domains. The N-terminal domain (1–83) is rendered in yellow and blue, with one dimer is yellow and the other one is blue. The C-terminal domains (87–229) are rendered in salmon. The flexible regions are presented as magenta dots and include N-termini of A46(1–83), C-termini of A46(87–229) and the linker between A46(1–83) and A46(87–229).</p

Structural Studies of an Anti-Inflammatory Lectin from <i>Canavalia boliviana</i> Seeds in Complex with Dimannosides

<div><p>Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the <i>Diocleinae</i> subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from <i>Canavalia boliviana</i> (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using <i>in vivo</i> and <i>in vitro</i> models.</p></div

The pH-dependent oligomerization of ConA and Cbol determined with SAXS.

<p>The pH-dependent oligomerization of ConA and Cbol determined with SAXS.</p

Chemotaxis assay of human neutrophils incubated in the presence of Cbol.

<p>Cells were allowed to migrate in the Boyden chamber toward the medium alone or IL-8. Cells were pre incubated with different concentrations of CboL. Data show means ± SD from three independent experiments performed in triplicate.</p

Proposed amino acid sequence of Cbol.

<p>Assemble from sequences of degradation products generated by cleavages with trypsin (T-), chymotrypsin (Q-) and pepsin (P-). The letter X in Cbol sequence represents residues of leucine or isoleucine, which cannot be distinguished by mass.</p