Binding of GSH at the active site of mLTC4S.

<p>A. Electron density 2fo-fc map contoured at 1.0 σ around GSH with Arg104 coordinating the sulfur in GSH. B. GSH bound at the active site, coordinated by several amino acids where the Arg51 - Tyr50 (indicated with a line) interaction in the human enzyme, is lost in the mLTC4S, which has a Phe in position 50.</p

Positional shift of Arg51 and loss of salt bridge at the active site of mLTC4S.

<p>A. Close up of the mLTC4S complex with SO₄²⁻, showing a shift in the position of Arg51 due to Phe50Tyr exchange. Human LTC4S is colored in green and mLTC4S is colored in gray. GSH is shown as green “lines”. *indicates that it is positioned on the neighboring subunit. B. Trimer of mLTC4S showing the amino acid exchange at position 50 where Phe in mLTC4S fails to make a salt bridge with Arg51. In hLTC4S, the Tyr50-Arg51 couple will likely contribute to trimer stability.</p

Steady state kinetic parameters of mLTC4S and hLTC4S against GSH and LTA₄.

<p>The enzyme activity was measured in 25mM Tris (pH 7.8), 0.1M NaCl, 0.05% DDM in the presence of either 30 µM LTA₄ and/or 5 mM GSH with 0.1 µg of enzyme.</p><p>**<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096763#pone.0096763-RinaldoMatthis2" target="_blank">[29]</a>.</p

Comparison of human and mouse LTC4S enzymes.

<p>A. Amino acid sequence alignment of human and mouse LTC4S generated with the program ClustalW. Species differences are highlighted in white. B. Mapping the amino acid differences (in red) between mouse and human trimeric LTC4S structures. The active site in one monomer is depicted with a bound GSH (green). In blue is the Phe50Tyr exchange positioned close to the active site.</p

The LTC₄ synthase reaction.

<p>A. Schematic drawing of the catalytic reaction of LTC4S where the allylic epoxide LTA₄ is conjugated with GSH at C6, to form LTC₄. B. Structure of the product analog S-hexyl GSH.</p

TK04 is a nanomolar competitive inhibitor of LTC4S.

<p>A. Chemical structure of TK04, the inhibitor used in this study. Dose-response curves for inhibition of mouse and human LTC4S by TK04. 100% activity corresponds to the enzyme activity without inhibitor, which was 44.0 µmol min⁻¹ mg⁻¹ for the mouse enzyme (red line) and 69.7 µmol min⁻¹ mg⁻¹ for the human enzyme (black line). The concentrations of substrates GSH and LTA₄ used in the assay were 5 mM and 20 µM, respectively. The IC₅₀ for mLTC4S was 135±30 nM and for the hLTC4S it was 134±16 nM.</p

Binding of S-hexyl GSH to the active site of mLTC4S.

<p>A. The trimeric form of mLTC4S with three bound S-hexyl GSH. B. Electron density 2fo-fc map, contoured at 1.0 σ around S-hexyl GSH. C. The hydrophobic cavity with S-hexyl GSH bound (yellow stick carbons) in the hydrophobic cleft. Amino acids facing the cavity are from monomers A (yellow) and B (green).</p

Additional file 1 of Discovery of a small molecule that inhibits Bcl-3-mediated cyclin D1 expression in melanoma cells

Additional file 1: Supplementary Figure 1

The dimeric 5LO does not bind to nanodiscs or separate into monomeric 5LO.

<p>Native PAGE analysis of purified ND (Lane 1) and purified dimer 5LO (Lane 2). Dimeric 5LO incubated with ND with Ca²⁺ (Lane 3) shows only bands corresponding to the dimer (158 kDa) and the ND (260 kDa). Dimer 5LO incubated with ND but without Ca²⁺ (Lane 4) shows the same bands as for lane 3 indicating that the dimer is not split into monomers by the presence of a membrane.</p

5LO initial velocity and product formation.

<p>Initial velocities (column V_init) and total 5-HPETE/5-HETE formation (column Final Product) were determined by UV spectroscopy at 235 nm, during 5 min incubations with different activating factors. Ratio 5LO:ND is 1.3:1. Data are mean±SEM (n = 3). LTA₄ formation (column LT) in 10 min incubations with different activating factors was measured by LC-MS/MS. * Not detected. Data are mean±SEM(n = 2).</p