The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc..
The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of 125I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys63-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free 125I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration (Km = 44 ± 6 nm; kcat = 0.020 ± 0.007 s-1) and substrate inhibition above 0.5 μm (Ki = 2.5 ± 1.3 μm) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin-binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 (Ki = 2.0 ± 0.5 μm), consistent with the presence of the second E2-binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high-affinity Site 1 (Ki = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 (Kact = 750 ± 260 nm). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrate that the cryptic Site 1 is associated with thioester formation, whereas binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca2+-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin-binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2017 |
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| Erschienen: |
2017 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:292 |
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| Enthalten in: |
The Journal of biological chemistry - 292(2017), 47 vom: 24. Nov., Seite 19521-19536 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Todaro, Dustin R [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 23.01.2018 Date Revised 05.02.2021 published: Print-Electronic PDB: 3JW0 Citation Status MEDLINE |
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| doi: |
10.1074/jbc.M117.817882 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM27641831X |
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| 100 | 1 | |a Todaro, Dustin R |e verfasserin |4 aut | |
| 245 | 1 | 4 | |a The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly |
| 264 | 1 | |c 2017 | |
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| 500 | |a Date Completed 23.01.2018 | ||
| 500 | |a Date Revised 05.02.2021 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a PDB: 3JW0 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. | ||
| 520 | |a The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of 125I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys63-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free 125I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration (Km = 44 ± 6 nm; kcat = 0.020 ± 0.007 s-1) and substrate inhibition above 0.5 μm (Ki = 2.5 ± 1.3 μm) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin-binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 (Ki = 2.0 ± 0.5 μm), consistent with the presence of the second E2-binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high-affinity Site 1 (Ki = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 (Kact = 750 ± 260 nm). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrate that the cryptic Site 1 is associated with thioester formation, whereas binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca2+-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin-binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a E3 ubiquitin ligase | |
| 650 | 4 | |a enzyme kinetics | |
| 650 | 4 | |a enzyme mechanism | |
| 650 | 4 | |a linkage specificity | |
| 650 | 4 | |a polyubiquitin chain | |
| 650 | 4 | |a protein-protein interaction | |
| 650 | 4 | |a thiol exchange | |
| 650 | 4 | |a ubiquitin | |
| 650 | 4 | |a ubiquitylation (ubiquitination) | |
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| 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 Ubiquitin-Protein Ligases |2 NLM | |
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| 650 | 7 | |a SY7Q814VUP |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 292(2017), 47 vom: 24. Nov., Seite 19521-19536 |w (DE-627)NLM000004995 |x 1083-351X |7 nnns |
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