Ubiquitylation‐dependent oligomerization regulates activity of Nedd4 ligases

• Published in: The EMBO journal Vol. 36; no. 4; pp. 425 - 440
• Main Authors: Attali, Ilan, Tobelaim, William Sam, Persaud, Avinash, Motamedchaboki, Khatereh, Simpson‐Lavy, Kobi J, Mashahreh, Bayan, Levin‐Kravets, Olga, Keren‐Kaplan, Tal, Pilzer, Inbar, Kupiec, Martin, Wiener, Reuven, Wolf, Dieter A, Rotin, Daniela, Prag, Gali
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 15.02.2017; Blackwell Publishing Ltd; John Wiley and Sons Inc
• Subjects: Binding sites; Bioinformatics; Cellular biology; EMBO31; Endosomal Sorting Complexes Required for Transport - metabolism; Enzymes; Humans; Inactivation; Microfilament Proteins - chemistry; Microfilament Proteins - metabolism; Mutation; Nedd4; Nedd4 Ubiquitin Protein Ligases; oligomerization; Potassium Channels, Voltage-Gated - chemistry; Potassium Channels, Voltage-Gated - metabolism; Proteasome Endopeptidase Complex - chemistry; Proteasome Endopeptidase Complex - metabolism; Protein Multimerization; Receptor, Fibroblast Growth Factor, Type 1 - chemistry; Receptor, Fibroblast Growth Factor, Type 1 - metabolism; Rsp5; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; ubiquitylation; Yeasts; Rsp5; oligomerization; ubiquitylation; inactivation; Nedd4
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201694314

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 I KS 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 I KS channel, thus confirming the functional importance of E3‐ligase autoinhibition. Synopsis Nedd4 ubiquitin ligase auto‐modification represents a novel allosteric regulatory mechanism, in which HECT E3 trimerization after self‐ubiquitylation leads to reversible inactivation and stabilization of their numerous cellular substrates. Interaction of Nedd4‐conjugated ubiquitin with a HECT domain ubiquitin‐binding site allosterically opens a cryptic oligomerization interface. Ubiquitylated Nedd4 oligomerizes into ligase‐inactive trimers and hexamers. Expression of non‐ubiquitylatable ligase mutants causes unrestrained downregulation of substrate proteins in vitro and in vivo . Nedd4 ligase regulation by auto‐modification‐mediated oligomerization is conserved from yeast to human. Graphical Abstract Ubiquitin ligase auto‐modification results in reversible inactivation rather than proteasomal degradation in a HECT‐domain E3 family.