Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation]
Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas
The American Society for Biochemistry and Molecular Biology (ASBMB)
Published 2018
The American Society for Biochemistry and Molecular Biology (ASBMB)
Published 2018
| Publication Date: |
2018-11-24
|
|---|---|
| Publisher: |
The American Society for Biochemistry and Molecular Biology (ASBMB)
|
| Print ISSN: |
0021-9258
|
| Electronic ISSN: |
1083-351X
|
| Topics: |
Biology
Chemistry and Pharmacology
|
| Published by: |
| _version_ | 1836399091063455744 |
|---|---|
| autor | Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas |
| beschreibung | 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. |
| citation_standardnr | 6360321 |
| datenlieferant | ipn_articles |
| feed_id | 43 |
| feed_publisher | The American Society for Biochemistry and Molecular Biology (ASBMB) |
| feed_publisher_url | http://www.asbmb.org/ |
| insertion_date | 2018-11-24 |
| journaleissn | 1083-351X |
| journalissn | 0021-9258 |
| publikationsjahr_anzeige | 2018 |
| publikationsjahr_facette | 2018 |
| publikationsjahr_intervall | 7984:2015-2019 |
| publikationsjahr_sort | 2018 |
| publisher | The American Society for Biochemistry and Molecular Biology (ASBMB) |
| quelle | Journal of Biological Chemistry |
| relation | http://feedproxy.google.com/~r/jbc/SUcv/~3/So_Cl_TqElY/18192.short |
| search_space | articles |
| shingle_author_1 | Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas |
| shingle_author_2 | Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas |
| shingle_author_3 | Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas |
| shingle_author_4 | Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas |
| shingle_catch_all_1 | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] 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. Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas The American Society for Biochemistry and Molecular Biology (ASBMB) 0021-9258 00219258 1083-351X 1083351X |
| shingle_catch_all_2 | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] 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. Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas The American Society for Biochemistry and Molecular Biology (ASBMB) 0021-9258 00219258 1083-351X 1083351X |
| shingle_catch_all_3 | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] 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. Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas The American Society for Biochemistry and Molecular Biology (ASBMB) 0021-9258 00219258 1083-351X 1083351X |
| shingle_catch_all_4 | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] 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. Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, Arthur L. Haas The American Society for Biochemistry and Molecular Biology (ASBMB) 0021-9258 00219258 1083-351X 1083351X |
| shingle_title_1 | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] |
| shingle_title_2 | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] |
| shingle_title_3 | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] |
| shingle_title_4 | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] |
| timestamp | 2025-06-30T23:37:27.749Z |
| titel | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] |
| titel_suche | Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly [Protein Synthesis and Degradation] |
| topic | W V |
| uid | ipn_articles_6360321 |