Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly
© 2018 Todaro et al..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2018 |
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| Erschienen: |
2018 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:293 |
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| Enthalten in: |
The Journal of biological chemistry - 293(2018), 47 vom: 23. Nov., Seite 18192-18206 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Todaro, Dustin R [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 24.06.2019 Date Revised 21.03.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1074/jbc.RA118.003716 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM289230675 |
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| 100 | 1 | |a Todaro, Dustin R |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly |
| 264 | 1 | |c 2018 | |
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| 500 | |a Date Completed 24.06.2019 | ||
| 500 | |a Date Revised 21.03.2021 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2018 Todaro et al. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
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| 650 | 4 | |a E3 | |
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| 650 | 4 | |a HECT ligase | |
| 650 | 4 | |a enzyme kinetics | |
| 650 | 4 | |a enzyme mechanism | |
| 650 | 4 | |a linkage specificity | |
| 650 | 4 | |a oligomer | |
| 650 | 4 | |a protein complex | |
| 650 | 4 | |a protein degradation | |
| 650 | 4 | |a protein-protein interactions | |
| 650 | 4 | |a transthiolation | |
| 650 | 4 | |a ubiquitin | |
| 650 | 4 | |a ubiquitin ligase | |
| 650 | 7 | |a Ubiquitin |2 NLM | |
| 650 | 7 | |a Polyubiquitin |2 NLM | |
| 650 | 7 | |a 120904-94-1 |2 NLM | |
| 650 | 7 | |a Nedd4 Ubiquitin Protein Ligases |2 NLM | |
| 650 | 7 | |a EC 2.3.2.26 |2 NLM | |
| 650 | 7 | |a Nedd4L protein, human |2 NLM | |
| 650 | 7 | |a EC 2.3.2.26 |2 NLM | |
| 650 | 7 | |a UBE3A protein, human |2 NLM | |
| 650 | 7 | |a EC 2.3.2.26 |2 NLM | |
| 650 | 7 | |a Ubiquitin-Protein Ligases |2 NLM | |
| 650 | 7 | |a EC 2.3.2.27 |2 NLM | |
| 700 | 1 | |a Augustus-Wallace, Allison C |e verfasserin |4 aut | |
| 700 | 1 | |a Klein, Jennifer M |e verfasserin |4 aut | |
| 700 | 1 | |a Haas, Arthur L |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t The Journal of biological chemistry |d 1945 |g 293(2018), 47 vom: 23. Nov., Seite 18192-18206 |w (DE-627)NLM000004995 |x 1083-351X |7 nnns |
| 773 | 1 | 8 | |g volume:293 |g year:2018 |g number:47 |g day:23 |g month:11 |g pages:18192-18206 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1074/jbc.RA118.003716 |3 Volltext |
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