NEDD4L intramolecular interactions regulate its auto and substrate Na V 1.5 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 _V 1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For Na _V 1.5, 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 _V 1.5. 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 _V 1.5. 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 _V 1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding Na _V 1.5 ubiquitination.
Competing Interests: Conflict of interest The authors declare no conflicts of interest in regards to this manuscript. S. B. G. is a cofounder and equity holder in the company Advanced Molecular Sciences, LLC. S. B. G. has been or is a consultant for Scorpion Therapeutics and Xinthera. P. A. C. has been a consultant for Scorpion Therapeutics.
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