Nedd4-2 is a Functional Oligomer Exhibiting Cooperative Allosteric Kinetics

Abstract

The Hect ligase Standard Model posits binding of the E2∼ubiquitin co-substrate to a single site within a monomeric Hect domain followed by transthiolation to form a Hect∼ubiquitin thioester intermediate. Transfer of the Hect-bound ubiquitin to lysyl residues on target proteins is then catalyzed by the ligase, processesively assembling polyubiquitin chains by distal addition. Recently, we demonstrated that the Hect ligase Nedd4-2 catalyzes polyubiquitin chain assembly by cooperative allosteric kinetics ([S]1/2=93±20 nM; kcat=0.067±0.008 s−1; nH=1.9±0.6) and exhibits substrate inhibition above 0.5 µM Ubc5B∼125I-ubiquitin, requiring the enzyme to function as an oligomer with two ordered E2∼ubiquitin binding sites of different affinities. These data support an alternative model for Hect ligase conjugation and are in agreement with our previous observations for E6AP. The current studies employ kinetic and biophysical methods to further explore the mechanism of Nedd4-2. Gel filtration chromatography and dynamic light scattering are consistent with trimerization. Truncation of the N-terminus yielding GST-Nedd4-2HECT (residues 597-955) reveals cooperative kinetics for polyubiquitin chain assembly (nH=2.6±1) with a ca. 104-fold reduction in kcat (1.9±0.2 x 10−6 s−1), demonstrating allostery intrinsic to the Hect domain and N-terminal contributions supporting oligomerization. Processing of GST yields monomeric Nedd4-2HECT resulting in monoubiquitination with hyperbolic kinetics (KM=39±7 nM; kcat=6.0±0.2 x 10−6 s−1) and loss of polyubiquitin chain assembly and substrate inhibition. Addition of Nedd4-2ΔHECT (residues 1-596) to wild type Nedd4-2 quantitatively inhibits activity; however, addition of the dominant negative Nedd4-2C922A mutant inhibits Nedd4-2 to 30% limiting activity, requiring the two sites to function in trans. These data support a model requiring trimerization and intersubunit interactions for polyubiquitin chain assembly consistent with our model of Proximal Indexation. [Supported by GM34009 to A.L.H.]