NEDD4L intramolecular interactions regulate its auto and substrate Na1.5 ubiquitination.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, Na1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For Na1.5, this process is critical as alterations in Na current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of Na1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of Na1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na current reduction using the NEDD4L HECT domain as cargo of a Na1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding Na1.5 ubiquitination.