Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination

Robert A. Chong, Kenneth Wu, Donald E. Spratt, Yingying Yang, Chan Lee, Jaladhi Nayak, Ming Xu, Rana Elkholi, Inger Tappin, Jessica Li, Jerard Hurwitz, Brian D. Brown, Jerry Edward Chipuk, Zhijian J. Chen, Roberto Sanchez, Gary S. Shaw, Lan Huang, And Zhen Qiang Pan

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF) βTrCP E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant UbR54A/Y59A diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by UbR54A/Y59A or UbK48R yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.

Original languageEnglish
Pages (from-to)8434-8439
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number23
StatePublished - 10 Jun 2014


  • E2 ubiquitin-conjugating enzyme
  • E3 ubiquitin ligase
  • Receptor ubiquitin


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