STRUCTURAL INSIGHTS INTO POLYUBIQUITIN CHAIN FORMATION MEDIATED BY NEDD4 HECT LIGASE
NEDD4 is the founding member of a family of HECT E3 ubiquitin ligases which regulates the trafficking and stability of receptors and signaling proteins. Precise spatiotemporal control of its activity is essential for orchestrating cellular functions. In resting condition, NEDD4 N-terminus by interacting with its C-terminal HECT catalytic domain maintains the E3 in auto-inhibited state. Activating stimuli, such as NEDD4 phosphorylation, prompt the release of its closed conformation enabling substrate ubiquitination. From the catalytic point-of-view, NEDD4 can modify substrates with a single ubiquitin moiety or K63-linked polyubiquitin chains. Despite its critical role, the structural and mechanistic aspects of NEDD4-mediated polyubiquitination remain enigmatic. Our lab previously identified a ubiquitin exosite within HECT as critical determinant for enzyme processivity. Here, we unveil that the ubiquitin exosite does not act as allosteric activator but likely stabilizes a nascent polyubiquitin chain towards the active site. Leveraging in-silico modeling and a novel TR-FRET-based ubiquitination assay, we pinpoint conserved HECT residues crucial for catalysis. Additionally, we dissected multiple determinants in the N-terminal domains of NEDD4 essential for intramolecular autoinhibition. Moreover, the establishment of a robust substrate-based TR-FRET assay revealed an additional layer of regulation for NEDD4 activation, which is mediated by the substrate itself. In parallel, we undertook cryo-electron microscopy approach to formally elucidate NEDD4 autoinhibition mechanism at the structural level. As of now, we have obtained an initial 9.4Å resolution density map, revealing both the HECT and C2 domains. In combination, these multifaceted approaches provide invaluable insights into the intricate mechanisms governing NEDD4-mediated polyubiquitination.
Ubiquitin and ubiquitination, E3 ligase, Cell signaling, Crystallography, Cryo-electron microscopy, Protein structure, Biochemistry, Chemistry, Fluorescence resonance energy transfer (FRET), Settore BIO/10 - Biochimica
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