Identification of conjugation specificity determinants unmasks vestigial preference for ubiquitin within the NEDD8 E2.
Ubiquitin-like proteins (UBLs) modify targets via related E1-E2-E3 cascades. How is UBL conjugation fidelity established? Here we report the basis for UBL selection by UBL conjugating enzyme 12 (Ubc12), which is specific for the neural precursor cell expressed, developmentally down-regulated protein 8 (NEDD8), and does not form a thioester-linked conjugate with ubiquitin. We systematically identified Ubc12 surfaces impeding Ubc12 approximately ubiquitin conjugate formation and found that several structurally dispersed E1 binding elements, rather than UBL-interacting surfaces, determine E2 approximately UBL specificity. In addition to roles for conserved E1 and E2 domains, unique structures contribute UBL specificity to the NEDD8 and ubiquitin pathways. By removing surface elements, without substituting corresponding sequences from ubiquitin E2s, we unmasked Ubc12's vestigial preference for ubiquitin over NEDD8 by approximately 10(10)-fold. This has implications for the evolution of specific functions among ubiquitin E2s. We also find that Ubc12 sequences dictating UBL selection map to the E3 binding site, thus providing a molecular mechanism preventing inappropriate modification of targets.
Huang
Danny T
DT
Howard Hughes Medical Institute, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38105, USA.
Zhuang
Min
M
Ayrault
Olivier
O
Schulman
Brenda A
BA
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D044842:Cullin Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D000075687:NEDD8 Protein; D025801:Ubiquitin / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*
D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D050505:Mutant Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C471538:UBA1 protein, human; D025801:Ubiquitin; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes