WEHI Special Structural Biology Seminar hosted by Associate Professor Jacqui Gulbis

Oliver Clarke PhD
Assistant Professor of Physiology and Cellular Biophysics (in Anesthesiology and the Irving Institute for
Clinical and Translational Research), Columbia University Medical Center, Columbia University, USA

Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies

Davis Auditorium

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Including Q&A session

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins— ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis— the E2 binding site, the catalytic cysteine, and the ubiquitin exosite— as revealed by a 3.1 Å cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and CaV2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.

All welcome!