Characterization of the HECT Domain Ubiquitin Ligase Nedd4‐2

Nedd4‐2 is a WW domain‐containing HECT (Homologous to E6AP C‐Terminus) ligase best known for its role in targeting cell surface receptors, including ENaC in the distal kidney, for Lys 63 ‐linked polyubiquitin chain‐mediated endocytic uptake and lysosomal degradation. The Nedd4‐like ligases, including Nedd4‐2, also function in the pathway for viral budding of Ebola and Marburg viruses, among others. By employing rates of 125 I‐polyubiquitin chain assembly as a functional readout of ligase activity, we quantitatively explore the mechanism of Nedd4‐2 in biochemically‐defined assays. We demonstrate that Nedd4‐2 exhibits cooperative allosteric kinetics (n=1.8±0.7) with varying Ubc5B~ 125 I‐ubiquitin thioester substrate concentrations ([S] 1/2 =103±29 nM; k cat =0.07±0.01 s −1 ), requiring a priori that the enzyme exist as an oligomer. Gel filtration analysis reveals that the active form of Nedd4‐2 migrates as a trimer of 250 kDa, larger than the predicted 110 kDa monomer, confirmed by dynamic light scattering. Successive addition of a dominant negative Nedd4‐2C922A active site mutant inhibits wild type Nedd4‐2 to limiting 30% activity, consistent with polyubiquitin chain assembly requiring intersubunit interactions within an intact trimer. Based on sequence and structural homology of the predicted Nedd4‐2 trimer to E6AP, we identify Glu 646 and Arg 604 as conserved putative catalytic residues by point mutagenesis. Our studies demonstrate Ca 2+ ‐independent polyubiquitin chain assembly, contrary to previous reports of Ca 2+ ‐dependent C2 domain inhibition of Nedd4‐2 activity. These findings led us to further investigate the role of the N‐terminal C2 domain on Nedd4‐2‐catalyzed chain assembly. Truncation of the C2 domain of Nedd4‐2 to yield Nedd4‐2ΔC2 leads to a 2‐4‐fold increase in k cat and loss of cooperativity. Exogenous C2 domain non‐competitively inhibits Nedd4‐2ΔC2 (K i =68±0.93 μM) without restoring cooperativity, consistent with a modest Ca 2+ ‐independent allosteric autoinhibition by the C2 domain via intersubunit interactions. Using the proteasomal subunit S5a as a model substrate in vitro, we demonstrate that Nedd4‐2 catalyzes S5a conjugation with apparent hyperbolic kinetics (K M =254±0.078 nM; k cat = 0.020±0.002 s −1 ), requiring the polyubiquitin chain assembly and substrate conjugation half reactions to be conformationally independent. The good agreement between k cat for S5a conjugation (0.020±0.002 s −1 ) and that for free polyubiquitin chain assembly (0.07±0.01 s −1 ) suggests that Nedd4‐2 does not discriminate between lysine donors and that both conjugation half reactions share similar transition states. Parallels to our recent observations with E6AP suggest mechanistic conservation with subtle differences based on distinct sequence and structural variations for Nedd4‐2. These findings support mechanistic conservation within the HECT ligase superfamily. Support or Funding Information [Supported by GM034009 to A.L.H]