Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases

  • Nat Struct Mol Biol. 2024 Feb;31(2):378-389. doi: 10.1038/s41594-023-01206-1.
Joanna Liwocha  #  1 ,  Jerry Li  #  2 ,  Nicholas Purser  2 ,  Chutima Rattanasopa  2 ,  Samuel Maiwald  1 ,  David T Krist  1 ,  Daniel C Scott  3 ,  Barbara Steigenberger  4 ,  J Rajan Prabu  1 ,  Brenda A Schulman  5 ,  Gary Kleiger  6  7
Affiliations
  • 1. Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany.
  • 2. Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV, USA.
  • 3. Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • 4. Mass Spectrometry Core Facility, Max Planck Institute of Biochemistry, Martinsried, Germany.
  • 5. Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany. [email protected].
  • 6. Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany. [email protected].
  • 7. Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV, USA. [email protected].
  • # Contributed equally.
PMID: 38326650 DOI: 10.1038/s41594-023-01206-1
Abstract

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating Enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING Ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.

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