TY - JOUR
T1 - Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases
AU - Attali, Ilan
AU - Tobelaim, William Sam
AU - Persaud, Avinash
AU - Motamedchaboki, Khatereh
AU - Simpson-Lavy, Kobi J.
AU - Mashahreh, Bayan
AU - Levin-Kravets, Olga
AU - Keren-Kaplan, Tal
AU - Pilzer, Inbar
AU - Kupiec, Martin
AU - Wiener, Reuven
AU - Wolf, Dieter A.
AU - Rotin, Daniela
AU - Prag, Gali
N1 - Publisher Copyright:
© 2017 The Authors
PY - 2017/2/15
Y1 - 2017/2/15
N2 - Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.
AB - Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.
KW - Nedd4
KW - Rsp5
KW - inactivation
KW - oligomerization
KW - ubiquitylation
UR - http://www.scopus.com/inward/record.url?scp=85008474012&partnerID=8YFLogxK
U2 - 10.15252/embj.201694314
DO - 10.15252/embj.201694314
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AN - SCOPUS:85008474012
SN - 0261-4189
VL - 36
SP - 425
EP - 440
JO - EMBO Journal
JF - EMBO Journal
IS - 4
ER -