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Mechanistic Characterization of the HECT Domain Ubiquitin Ligase Nedd4‐2
Author(s) -
Todaro Dustin,
AugustusWallace Allison,
Haas Arthur
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.883.10
Subject(s) - nedd4 , ubiquitin ligase , ubiquitin , chemistry , trimer , enzyme kinetics , biophysics , biochemistry , dna ligase , kinetics , stereochemistry , microbiology and biotechnology , enzyme , active site , biology , dimer , physics , organic chemistry , quantum mechanics , gene
Nedd4‐2 is a WW‐domain containing HECT ligase responsible for the ubiquitination of a broad range of cell surface receptors, targeting them for endocytic uptake and degradation. The Nedd4‐like ligases, including Nedd4‐2, have also been implicated in facilitating viral budding of Ebola and Marburg viruses, among others. By employing rates of 125 I‐polyubiquitin chain formation as a functional readout of ligase activity, we quantitatively explore the mechanism and properties of Nedd4‐2. We demonstrate that Nedd4‐2 exhibits cooperative kinetics (n=2.0±0.4) with respect to varying Ubc5B~ 125 I‐ubiquitin thioester substrate concentrations (K M =160±84 nM; k cat =0.092±0.002 s ‐1 ) and substrate inhibition at micromolar concentrations (K i =1.5±0.46 μM), the latter requiring ordered binding at two functionally distinct sites. The product analog Ubc5BC85A displays non‐competitive inhibition (K i =1.4±0.26 μM), consistent with the two site model. Observation of cooperative kinetics requires that Nedd4‐2 exist as an oligomer. Static light scattering demonstrates that Nedd4‐2 forms a trimer (MW=250 kDa), in good agreement with the predicted molecular weight of 330 kDa. Mutation of Phe 823 within the conserved hydrophobic pocket known to mediate trimerization of E6AP completely abrogates Nedd4‐2 catalytic activity. The phenylalanine derivative Ac‐Phe‐NH 2 also inhibited catalytic activity non‐competitively (K i =30±10 mM), presumably by disrupting Phe 823 binding within the hydrophobic pocket to destabilize the enzyme. Parallels with our recent observations reported for E6AP indicate these structural and catalytic properties are likely conserved within the HECT ligase superfamily. [Supported by GM034009 to A.L.H.]