NEDD4L intramolecular interactions regulate its auto and substrate Na_V1.5 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, Na_V1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For Na_V1.5, 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 Na_V1.5. 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 Na_V1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na⁺ current reduction using the NEDD4L HECT domain as cargo of a Na_V1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding Na_V1.5 ubiquitination.