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>