cted by NAE can form thioester conjugates with NAE and Ubc12. We synthesized peptides corresponding to the last seven residues of the UB variants N1, N7, N11, N21, N26, N27, and N30 which are referred to as pN1, pN7, pN11 and so on. The full-length proteins of these UB variants all showed similar activities with NAE as wt Nedd8 in the ATP-PPi exchange reaction. We assayed the ATP-PPi exchange activity of the peptides with NAE 
and found that peptides pN1, pN7 and pN26, derived from the C-terminal Nedd8-Like Ubiquitin Variants sequences of UB variants N1, N7 and N26, can be activated at a level significantly higher than the background with no peptide added. Kinetic analysis of the three peptides suggested that the peptides have similar kcat values in the range of 23 26 min21, comparable to that of the wt Nedd8 protein with a kcat of 55 min21. In contrast the peptides have K1/2 between 88 155 mM, which are 100-fold higher than full-length Nedd8 suggesting that other interfaces between UB and NAE significantly contribute to NAE recognition besides the C-terminal peptides of the UB variants. Peptide pN26 has the native C-terminal sequence of wt Nedd8, documenting that the C-terminal peptide of Nedd8 alone has substantial activity with NAE. We next conjugated biotin to the N-termini of the peptides pN1, pN7, and pN26 and found that all three peptides can form thioester conjugates with NAE based on the Western blot of the transfer reaction probed with streptavidin-HRP conjugate. We thus refer to these peptides as “Nedd8-mimicking peptides”since they can be activated by NAE and form thioester conjugates with NAE as the wt Nedd8 protein. However, only peptide pN26, with its sequence matching the C-terminus of native Nedd8, showed a strong activity for the R-7128 formation of peptide,Ubc12 conjugates and the peptide can be further transferred to cullin 3. In contrast peptide pN7 only has low activity for transferring to Ubc12 while pN1 showed no observable 17636045 formation of peptide,Ubc12 conjugates, though both Nedd8-Like Ubiquitin Variants Km Full-length proteins Nedd8 wt UB N1 N7 N11 N27 N26 pN1 pN7 pN26 71 76 kcat kcat/Km 1.360.2 ND 55612 ND 43 0.012 UB variants from phage selection 1.860.3 2.060.2 2.260.3 2.860.8 1.660.2 3062 3265 3466 2663 52616 16 16 16 9.0 32 Nedd8 mimicking peptides 130641 155650 88625 2464 2466 2667 0.18 0.16 0.29 ND, not determined because of low activity. doi:10.1371/journal.pone.0070312.t002 Nedd8 transferring cascade, the chase of the loading reaction with full-length Nedd8 did not produce any Nedd8 conjugates with NAE, Ubc12 or cullin. These results demonstrate that the Nedd8-mimicking peptides can be used as inhibitors to block cullin neddylation. Discussion UB C-terminal Residues for NAE and UAE Recognition peptides can be loaded on NAE at a similar level as pN26. These results indicate that 22634634 only the native Nedd8 Cterminus present in pN26 retains the ability to pass through the NAE-Ubc12 cascade for cullin modification. The non-native residues in pN1 and p27 may block the peptides from transferring to Ubc12 and cullin although they do not interfere with NAE recognition. Inhibiting the NAE-Ubc12 Cascade by the Nedd8 Mimicking Peptides We rationalized that if the Nedd8-mimicking peptides can be loaded on NAE and Ubc12, they should block Nedd8 transfer through the cascade since the peptides occupy the catalytic Cys residues of NAE and Ubc12 to prevent Nedd8 conjugation. These peptides can serve as road blocks to stop Nedd8 trans