Structural basis for UBA3’s Arg190s negative selectivity against a UBL’s Arg72. Superimposition of wild-type (21) and mutant APPBP1-UBA3-NEDD8 structures was performed using least-squares fitting over all atoms in O (33). UBA3’s residue 190 is shown in various shades of red; NEDD8’s residue 72 in yellow; nitrogen, blue; and oxygen, light red. (A) Overall superimposition of APPBP1-UBA3Arg190Gln (rose)-NEDD8Ala72Arg (melon, “ubiquitinized”) and APPBP1-UBA3Arg190Ala (maroon)-NEDD8Ala72Arg (chartreuse, “wild-type-opposite”) complexes, with close-up view around the NEDD8 mutant’s Arg72 and UBA3’s residue 190. (B) Close-up view showing NEDD8 mutant Arg72 from APPBP1-UBA3Arg190Gln-NEDD8Ala72Arg (melon) and from APPBP1-UBA3Arg190Ala-NEDD8Ala72Arg (chartreuse), and Arg190 (red) from wild-type APPBP1-UBA3-NEDD8. (C) Close-up view showing Arg190 (violet) and NEDD8 mutant Gln72 from APPBP1-UBA3Arg190 (wt)-NEDD8Ala72Gln, and Arg190 (red) from wild-type APPBP1-UBA3-NEDD8.