Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly
| Autor: | Dustin R. Todaro, Jennifer M. Klein, Allison C. Augustus-Wallace, Arthur L. Haas |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
HECT domain Nedd4 Ubiquitin Protein Ligases Ubiquitin-Protein Ligases Amino Acid Motifs macromolecular substances Protein degradation Biochemistry Catalysis 03 medical and health sciences Ubiquitin Catalytic Domain Monoubiquitination Humans Homology modeling Polyubiquitin Molecular Biology chemistry.chemical_classification DNA ligase Isopeptide bond biology Chemistry Cell Biology Ubiquitin ligase Kinetics 030104 developmental biology biology.protein Biophysics Enzymology |
| Zdroj: | The Journal of biological chemistry. 293(47) |
| ISSN: | 1083-351X |
| Popis: | 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 (125)I-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 ≥10(4)-fold decrease in k(cat) for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (K(i) = 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. |
| Databáze: | OpenAIRE |
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