Specificity of the E1-E2-E3 enzymatic cascade for ubiquitin C-terminal sequences identified by phage display

Bo Zhao, Karan Bhuripanyo, Jeffrey Schneider, Keya Zhang, Hermann Schindelin, David Boone, Jun Yin*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


Ubiquitin (UB) is a protein modifier that regulates many essential cellular processes. To initiate protein modification by UB, the E1 enzyme activates the C-terminal carboxylate of UB to launch its transfer through the E1-E2-E3 cascade onto target proteins. In this study, we used phage display to profile the specificity of the two human E1 enzymes, Ube1 and Uba6, toward the C-terminal sequence of UB ending with 71LRLRGG76. Phage selection revealed that while Arg72 of UB is absolutely required for E1 recognition, UB residues at positions 71, 73, and 74 can be replaced with bulky aromatic side chains, and Gly75 of UB can be changed to Ser, Asp, and Asn for efficient E1 activation. We have thus found that the E1 enzymes have substantial promiscuity regarding the UB C-terminal sequence. The UB variants from phage selection can also be transferred from E1 to E2 enzymes; however, they are blocked from further transfer to the E3 enzymes. This suggests that the C-terminal sequence of UB is important for its discharge from E2 and subsequent transfer to E3. In addition, we observed that the Leu73Phe and Leu73Tyr single mutants of UB are resistant to cleavage by deubiquitinating enzymes (DUBs), although they can be assembled by the E1-E2-E3 cascade into poly-UB chains, thus indicating differences in UB C-terminal specificities between the E1 and DUBs. Consequently these UB mutants may provide stability to UB polymers attached to cellular proteins and facilitate the elucidation of the biological signals encoded in the UB chains.

Original languageEnglish (US)
Pages (from-to)2027-2035
Number of pages9
JournalACS chemical biology
Issue number12
StatePublished - Dec 21 2012

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine


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