CSN6^ΔC binds to CSN5^ΔC.

(A) Human CSN6^ΔC displays a classical MPN fold. 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) CSN6^ΔC binding to CSN5^ΔC engages a conserved surface. CSN6 sequence conservation is displayed on the molecular surface of CSN6^ΔC with a colour coding blue to red from variable to conserved, respectively, as calculated with the PAT server [34]. The dotted line corresponds to the surface fingerprint of CSN5^{ΔC,Δ2–31,Δ232–257} in the docking model of CSN5^ΔC/CSN6^ΔC. (C) K_D values of CSN5^ΔC/CSN6^ΔC variant pairs obtained from ITC data. (D) Pro-Nedd8 processing by CSN5^ΔC. Time course assay of pro-Nedd8 (1 µM) processing by CSN5^ΔC,R106T/CSN6^ΔC (top; 4 nM, obtained from mixing 150 nM CSN5^ΔC,R106T and 200 nM CSN6^ΔC), CSN5^ΔC,R106T/CSN6^ΔC,H44A (middle; 4 nM) CSN5^ΔC,R106T/CSN6^ΔC,V115E (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^ΔC,WT in the presence of CSN6^ΔC displays robust activity on a range of substrates, as illustrated in Figure 1D,E,F, its level remains lower to that of CSN5^ΔC,R106T in the presence of CSN6^ΔC. 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.