The mechanism of Neural Precursor Cell Expressed Developmentally Down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly [Protein Synthesis and Degradation]

The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically-defined kinetic assays examining rates of 125I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys63-linked polyubiquitin chains. Full length Nedd4-2 catalyzes free 125I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration (KM = 44 ± 6 nM; kcat = 0.020 ± 0.007 s-1) and substrate inhibition above 0.5 μM (Ki = 2.5 ± 1.3 μM) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 (Ki = 2.0 ± 0.5 μM) consistent with the presence of the second E2 binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high affinity Site 1 (Ki = 720 ± 340 nM) and non-essential activation at the lower affinity Site 2 (Kact = 750 ± 260 nM). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrates that the cryptic Site 1 is associated with thioester formation, while binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca2+-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family.

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