CSN6^ΔC activates CSN5^ΔC.

<p>(<b>A</b>) <b>Studied protein fragments (brown)</b>. The MPN domain boundaries are indicated with dotted lines. (<b>B</b>) <b>Binding of CSN5^ΔC and CSN6^ΔC measured by ITC.</b> Titration experiments were carried out by injecting concentrated CSN6^ΔC solution into 100 µM CSN5 variants. (<b>C</b>) <b>K_D values of CSN5^ΔC/CSN6^ΔC pairs obtained from ITC data.</b> (<b>D</b>) <b>Activity towards Nedd8-AMC.</b> Nedd8-AMC hydrolysis rate plot versus substrate concentration (200 nM CSN5^ΔC alone; 4 nM CSN5^ΔC/CSN6^ΔC complex; 23 nM CSN), with data fitted using the Michaelis-Menten equation. Below: Associated kinetic parameters of the Nedd8-AMC cleavage derived from the left plot. In the CSN5^ΔC,R106T and CSN5^ΔC,WT/CSN6^ΔC experiments, substrate saturation conditions were not reached and k_app values were extracted from linear fitting of the k_obs versus substrate concentration data. (<b>E</b>) <b>Time-course deneddylation of Cullin1-Nedd8/Rbx1 (1 µM) by the CSN (4 nM; Top) or CSN5^ΔC,WT/CSN6 (4 nM; Bottom).</b> Samples from each time point were analysed on Coomassie stained 15% Tris-tricine SDS-PAGE. Quantification of the cullin deneddylation was carried out. The bands corresponding to Cullin1-Nedd8 and to Cullin1 show doublet that arise from Cullin1 degradation. (<b>F</b>) <b>Deneddylase activity on the Cullin1-Nedd8/Rbx1.</b> Substrate hydrolysis (1 µM) is shown as a function of time for 4 nM enzyme (CSN, triangle; CSN5^ΔC,WT/CSN6^ΔC, square). For context, CSN5^ΔC,R106T alone at 900 nM concentration produces less than 30% of deneddylated Cullin1 for 120 min reaction (data point not shown for clarity). Data information: N.D., not significant; -, parameters not measured; error bars = s.d.; experiments done at least twice.