CSN6ΔC activates CSN5ΔC.
(A) Studied protein fragments (brown). The MPN domain boundaries are indicated with dotted lines. (B) Binding of CSN5ΔC and CSN6ΔC measured by ITC. Titration experiments were carried out by injecting concentrated CSN6ΔC solution into 100 µM CSN5 variants. (C) KD values of CSN5ΔC/CSN6ΔC pairs obtained from ITC data. (D) Activity towards Nedd8-AMC. Nedd8-AMC hydrolysis rate plot versus substrate concentration (200 nM CSN5ΔC alone; 4 nM CSN5ΔC/CSN6ΔC 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 CSN5ΔC,R106T and CSN5ΔC,WT/CSN6ΔC experiments, substrate saturation conditions were not reached and kapp values were extracted from linear fitting of the kobs versus substrate concentration data. (E) Time-course deneddylation of Cullin1-Nedd8/Rbx1 (1 µM) by the CSN (4 nM; Top) or CSN5ΔC,WT/CSN6 (4 nM; Bottom). 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. (F) Deneddylase activity on the Cullin1-Nedd8/Rbx1. Substrate hydrolysis (1 µM) is shown as a function of time for 4 nM enzyme (CSN, triangle; CSN5ΔC,WT/CSN6ΔC, square). For context, CSN5ΔC,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.