Structure of NEDP1 alone and in complex with NEDD8. (A) Monomers of NEDP1 from the apo (wheat) and covalent complex (slate). The loop centred around residue 100 is shown in green and is profoundly changed in the complex. (B) The final 2F_o−F_c electron density different map of the linkage contoured at 1σ. (C) Stereo diagram of the NEDP1–NEDD8 complex. NEDP1 is coloured slate and NEDD8 pink. The residues mutated are shown and labelled. (D) The β-sheet-like interaction between nep1 and NEDD8.

Processing activities of NEDP1 mutant proteins. The substrate used in the assay is His-MBP-NEDD8-Ub and the assays are as detailed in Material and methods. NEDP1 and its mutants were mixed with the substrate and after incubation for 30 min at 37°C, the reaction was stopped by adding 6 × Laemmli loading buffer and boiled for 3 min. The processing products were fractionated using SDS–PAGE and the gel stained with 0.25% Coomassie blue. The designation of each mutant enzyme is indicated. CON: substrate only. The upper arrow indicates the substrate His-MBP-NEDD8-Ub, while the lower arrow indicates the cleavage product His-MBP-NEDD8. The released Ub has migrated off the bottom of the gel and is therefore not detected. Data presented are representative of three independent experiments.

How NEDP1 discriminates between NEDD8 and ubiquitin. (A) Superposition of NEDD8 and ubiquitin, the key change is the backbone centred on residue 72. (B) The NEDP1–NEDD8 complex is shown coloured as before. Ubiquitin is shown as an yellow ribbon. The ubiquitin is positioned based on a superposition of NEDP1 and Yuh1. It is clear that ubiquitin has a very different spatial relationship to its protease. Residue 72 is shown and labelled; it is the kink at this position that is responsible for the dramatic difference in orientation. (C) Time course of NEDP1 protein on processing wild-type NEDD8 and NEDD8 A/R mutant. NEDP1 enzyme was incubated with NEDD8 and A72R mutant proteins at 37°C and the reaction was stopped at 0.5, 1, 2, 3, 4 and 6 min by adding an equal volume of 2 × Laemmli loading buffer and boiling for 3 min. The His-MBP-N8 was fractionated by SDS–PAGE and quantified by densitometric analysis using a multianalysis system (Doc-2000, Bio-Rad). The data presented are the mean of three independent experiments. Error bars represent the 95% confidence of the calculated means. (D) Activities of NEDP1 and HAUSP on processing of ubiquitin and R72A ubiquitin. Substrates used in the assay are His-MBP-N8-UB, His-MBP-N8(m)-UB (NEDD8 A72R mutant), HisUB-UB and HisUB9(m)-UB (ubiquitin R72A mutant). Substrates were mixed with NEDP1 or HAUSP and incubated for 30 min at 37°C. The processing products were fractionated in SDS–PAGE and stained with 0.25% Coomassie blue.

Sequence alignment of NEDD8 with ubiquitin and SUMO and NEDP1 with Senp2 and ULP1. Sequences were aligned using ClustalW (Thompson et al, 1994). Conserved residues are shaded in grey. (A) Sequence alignment of NEDD8 with ubiquitin and SUMO. An alanine marked with an asterisk in NEDD8 was mutated to arginine, while an arginine in ubiquitin was mutated to alanine. Secondary structure elements (in yellow) above the sequence are indicated for NEDD8, while those (blue) below the sequences are indicated for ubiquitin. (B) Sequence alignment of NEDP1 with ULP1 and SENP2. Residues of the catalytic triad are highlighted in red. Residues involved in direct NEDD8 and NEDP1 intermolecular interaction are marked with an asterisk. Mutations in these residues were created by alanine substitution. Residues marked with a triangle were deleted in a loop deletion. The secondary structure elements (yellow) above the sequences are indicated for NEDP1 and those (blue) below the sequences are indicated for SENP2. Sequences shown are from NCBI protein databases: NEDD8 (NP_006147), ubiquitin (AAA36787), SUMO (AAH66306), NEDP1 (AAA36787), SENP2 (AAH40609) and ULP1 (1EUVA).

Comparison of protease-ubiquitin-like protein complexes. The complex between proteases, shown as electrostatic surfaces with the same scale in each and their target protein shown in a ribbon representation. Inset: The target turned to show its electrostatic surface that binds to the protease. (A) NEDP1–NEDD8. The protease interface is weakly acidic with no basic path. (B) Ulp1–SUMO. The strong positive patch at the bottom and a weaker smaller one at the top of protease are visible. (C) Yuh1–ubiquitin. The protease interface is much more acidic. In comparing ubiqutin and NEDD8, it can be seen that NEDD8 is less basic. This is consistent with changes in residues E53 and A72 (to Gly and Arg respectively in ubiquitin).

Ability of NEDP1 mutants to deconjugate NEDDylated p53 in vivo. Top panel: H1299 cells were transfected with expression constructs for p53, mdm2, 6His-NEDD8 and NEDP1 mutants as indicated. At 36 h after transfection, cells were lysed in guanidine hydrochloride and 6His-NEDD8-conjugated species purified on Ni-NTA agarose as described in Materials and methods. NEDDylated p53 was detected by Western blotting with DO-1 anti-p53 monoclonal antibody. Total levels of NEDP1 are shown in the bottom panel. Middle panel: As in top panel, but 6His-ubiquitin was cotransfected rather than 6His-NEDD8.