A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants

Mads Gabrielsen, Lori Buetow, Mark A Nakasone, Syed Feroj Ahmed, Gary J Sibbet, Brian O Smith, Wei Zhang, Sachdev S Sidhu, Danny T Huang

Research output: Contribution to journalArticlepeer-review

54 Citations (Scopus)
42 Downloads (Pure)

Abstract

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.

Original languageEnglish
Pages (from-to)456-470.e10
Number of pages26
JournalMolecular Cell
Volume68
Issue number2
DOIs
Publication statusPublished - 19 Oct 2017

Keywords

  • Drug Discovery/methods
  • Enzyme Activators/chemistry
  • Enzyme Inhibitors/chemistry
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Protein Multimerization/drug effects
  • Tumor Suppressor Proteins/agonists
  • Ubiquitin-Protein Ligase Complexes/antagonists & inhibitors
  • Ubiquitin-Protein Ligases
  • X-Linked Inhibitor of Apoptosis Protein/agonists

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