ts that other UB clones in the selected pool with substantial sequence variations can compete with the native C-terminal sequence of Nedd8 for NAE activation. Interestingly clone N11 with a C-terminal sequence 71AAAAGG76 was identified five times among all selected clones. Similar to N11, N52 with the Cterminal sequence of 71AAYAGG76 was also selected suggesting that Ala replacement of Leu and Arg at positions 71, 73 and 74 can still lead to NAE activation of the UB mutant. Thus, the native residues at these positions in UB or NAE should only have limited contributions to Nedd8 recognition by NAE. Modeling the Interaction between NAE and the Cterminal Sequences of UB Selected by Phage Display A comparison of the crystal structures of NAE and UAE in complex with Nedd8 and UB, respectively, showed that Nedd8 and UB bind to the E1 C.I. Natural Yellow 1 manufacturer enzymes in a similar mode . The C-terminal peptides of both Nedd8 and UB adopt an extended conformation so that the terminal Gly76 can approach the ATP molecule bound at the bottom of the adenylation domain of the E1 enzymes. The C-terminal peptides of Nedd8 and UB are 
also wrapped around by a crossover loop of their cognate E1 enzymes to guide their entrance into the ATP binding pocket. The side chains of Ala72 of Nedd8 and Arg72 of UB point into the same direction in the complex structure with the E1s. Ala72 of Nedd8 is docked in a well-defined binding pocket of limited space assembled by Asn188, Arg190, Tyr207 and Tyr321 of the Uba3 subunit of NAE. Similarly Uba1 residues Asn574, Gln576, Tyr586, and Asp591 constitute a larger pocket for specific recognition of Arg72 of UB. Except for Ala72 of Nedd8 and Arg72 of UB, other residues including Leu71, Leu73 and Arg74 at the Ctermini of Nedd8 and UB are identical, with Leu71 and Leu73 pointing to the opposite side of NAE and UAE while Arg74 is oriented in a nearly perpendicular direction to that of Ala72 of Nedd8 or Arg72 of UB. In case of all three residues, there is significantly more open space around their side chains compared to the residue at position 72. This might explain the enrichment of aromatic residues at positions 71, 73 and 74 when the UB library was selected with NAE in this study and selected with UAE in an earlier study. To further characterize the recognition of the C-terminal 25277138 sequences of UB variants by NAE, we transplanted the C-terminal peptides of UB variants N1, N7 and N27 onto the Nedd8 scaffold and modeled the binding of the Nedd8 mutants with NAE based on the crystal structure of Nedd8-NAE-ATP complex . The modeled structures showed that NAE can accommodate larger side chains such as Met, Phe, Tyr and Trp at positions 71, 73 and 74 at the C-terminus of Nedd8. Nedd8-Like Ubiquitin Variants The binding pocket of NAE for the wt Ala72 of Nedd8 is relatively limited in size, so Leu72 in N1 and N7 and Gln72 in N27 seem at first ill-suited to contribute to positive interactions with the NAE residues. However, a more detailed analysis reveals that both Leu72 and 3158656 Gln72 can be accommodated by the NAE binding pocket after subtle adjustment of their side chain conformations to avoid unfavorable contacts with nearby NAE residues. A crystal structure of the Ala72Gln mutant of Nedd8 in complex with NAE already demonstrated that Gln72 of Nedd8 can fit into the binding pocket of NAE for Ala72 recognition and engage in hydrogen bonding interactions with Arg190 of the Uba3 subunit of NAE. It has been reported that the Arg72Leu mutant of UB can be activated b