التفاصيل البيبلوغرافية
| العنوان: |
Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly. |
| المؤلفون: |
Todaro, Dustin R.1, Augustus-Wallace, Allison C.1, Klein, Jennifer M.1, Haas, Arthur L.1,2 ahaas@lsuhsc.edu |
| المصدر: |
Journal of Biological Chemistry. 11/23/2018, Vol. 293 Issue 47, p18192-18206. 15p. |
| مصطلحات موضوعية: |
*UBIQUITIN ligases, *GEL permeation chromatography, *BINDING sites, *OLIGOMERIZATION, *BIOPHYSICS, *HOMOLOGY (Biochemistry) |
| مستخلص: |
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±1.2mM) of polyubiquitin chain elongation bydestabilizingtheactivetrimer,suggestingamechanismfortherapeuticallytargetingHECTligases. Additionalkineticexperiments 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. [ABSTRACT FROM AUTHOR] |
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