Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly

J Biol Chem. 2018 Nov 23;293(47):18192-18206. doi: 10.1074/jbc.RA118.003716. Epub 2018 Oct 4.

Abstract

The NEDD4-2 (neural precursor cell-expressed developmentally down-regulated 4-2) HECT ligase catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two functionally distinct E2∼ubiquitin-binding sites, analogous to the trimeric E6AP/UBE3A HECT ligase. This conserved catalytic mechanism suggests that NEDD4-2, and presumably all HECT ligases, requires oligomerization to catalyze polyubiquitin chain assembly. To explore this hypothesis, we examined the catalytic mechanism of NEDD4-2 through the use of biochemically defined kinetic assays examining rates of 125I-labeled polyubiquitin chain assembly and biophysical techniques. The results from gel filtration chromatography and dynamic light-scattering analyses demonstrate for the first time that active NEDD4-2 is a trimer. Homology modeling to E6AP revealed that the predicted intersubunit interface has an absolutely conserved Phe-823, substitution of which destabilized the trimer and resulted in a ≥104-fold decrease in kcat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (Ki = 8 ± 1.2 mm) of polyubiquitin chain elongation by destabilizing the active trimer, suggesting a mechanism for therapeutically targeting HECT ligases. Additional kinetic experiments indicated that monomeric NEDD4-2 catalyzes only HECT∼ubiquitin thioester formation and monoubiquitination, whereas polyubiquitin chain assembly requires NEDD4-2 oligomerization. These results provide evidence that the previously identified sites 1 and 2 of NEDD4-2 function in trans to support chain elongation, explicating the requirement for oligomerization. Finally, we identified a conserved catalytic ensemble comprising Glu-646 and Arg-604 that supports HECT-ubiquitin thioester exchange and isopeptide bond formation at the active-site Cys-922 of NEDD4-2.

Keywords: E2; E3; HECT domain; HECT ligase; enzyme kinetics; enzyme mechanism; linkage specificity; oligomer; protein complex; protein degradation; protein-protein interactions; transthiolation; ubiquitin; ubiquitin ligase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Catalysis
  • Catalytic Domain
  • Humans
  • Kinetics
  • Nedd4 Ubiquitin Protein Ligases / chemistry*
  • Nedd4 Ubiquitin Protein Ligases / genetics
  • Nedd4 Ubiquitin Protein Ligases / metabolism*
  • Polyubiquitin / chemistry
  • Polyubiquitin / metabolism*
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • Ubiquitin
  • Polyubiquitin
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4L protein, human
  • UBE3A protein, human
  • Ubiquitin-Protein Ligases