10.1371/journal.pone.0105688.g002 Melissa Birol Melissa Birol Radoslav Ivanov Enchev Radoslav Ivanov Enchev André Padilla André Padilla Florian Stengel Florian Stengel Ruedi Aebersold Ruedi Aebersold Stéphane Betzi Stéphane Betzi Yinshan Yang Yinshan Yang François Hoh François Hoh Matthias Peter Matthias Peter Christian Dumas Christian Dumas Aude Echalier Aude Echalier CSN6<sup>ΔC</sup> binds to CSN5<sup>ΔC</sup>. Public Library of Science 2014 Cop 9 signalosome E 3 ubiquitin ligase family CSN 6 MPN CSN electron density map crl CSN 5 subunit CSN deneddylation activity cullin RING E 3 ubiquitin ligases CSN 5 CSN subunits isopeptidase activity 2014-08-21 04:22:05 Figure https://plos.figshare.com/articles/figure/_CSN6_916_C_binds_to_CSN5_916_C_/1148336 <p>(<b>A</b>) <b>Human CSN6<sup>ΔC</sup> displays a classical MPN fold</b>. The crystallised fragment of human CSN6 contains an MPN core (white ribbon), completed by a C-terminal extension (green ribbon). The asymmetric unit contains one molecule (A); the 2-fold crystallographic symmetry-related one, A′ is shown in beige as a surface. (<b>B</b>) <b>CSN6<sup>ΔC</sup> binding to CSN5<sup>ΔC</sup> engages a conserved surface.</b> CSN6 sequence conservation is displayed on the molecular surface of CSN6<sup>ΔC</sup> with a colour coding blue to red from variable to conserved, respectively, as calculated with the PAT server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105688#pone.0105688-Gracy1" target="_blank">[34]</a>. The dotted line corresponds to the surface fingerprint of CSN5<sup>ΔC,Δ2–31,Δ232–257</sup> in the docking model of CSN5<sup>ΔC</sup>/CSN6<sup>ΔC</sup>. (<b>C</b>) <b>K<sub>D</sub> values of CSN5<sup>ΔC</sup></b>/<b>CSN6<sup>ΔC</sup> variant pairs obtained from ITC data.</b> (<b>D</b>)<b> Pro-Nedd8 processing by CSN5<sup>ΔC</sup>.</b> Time course assay of pro-Nedd8 (1 µM) processing by CSN5<sup>ΔC,R106T</sup>/CSN6<sup>ΔC</sup> (top; 4 nM, obtained from mixing 150 nM CSN5<sup>ΔC,R106T</sup> and 200 nM CSN6<sup>ΔC</sup>), CSN5<sup>ΔC,R106T</sup>/CSN6<sup>ΔC,H44A</sup> (middle; 4 nM) CSN5<sup>ΔC,R106T</sup>/CSN6<sup>ΔC,V115E</sup> (bottom; 4 nM), were analysed on Coomassie stained SDS-PAGE gels. The time course assay was analysed on Coomassie stained 15% Tris-tricine SDS-PAGE. Quantification of pro-Nedd8 hydrolysis is specified, when possible, at the bottom of the gel. Quantification of the pro-Nedd8 processing was carried out as detailed in the Material and Methods section. Although CSN5<sup>ΔC,WT</sup> in the presence of CSN6<sup>ΔC</sup> displays robust activity on a range of substrates, as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105688#pone-0105688-g001" target="_blank">Figure 1D,E,F</a>, its level remains lower to that of CSN5<sup>ΔC,R106T</sup> in the presence of CSN6<sup>ΔC</sup>. For the pro-Nedd8 gel shift assay, as illustrated in this figure, for detection purpose, it was advantageous to use the best enzymatic system available.</p>