The Active Form of E6-associated protein (E6AP)/UBE3A Ubiquitin Ligase Is an Oligomer

• Published in: The Journal of biological chemistry Vol. 289; no. 2; pp. 1033 - 1048
• Main Authors: Ronchi, Virginia P., Klein, Jennifer M., Edwards, Daniel J., Haas, Arthur L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.01.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Angelman Syndrome - genetics; Angelman Syndrome - metabolism; Animals; Binding Sites - genetics; Biocatalysis; E3 Ubiquitin Ligase; Electrophoresis, Polyacrylamide Gel; Enzyme Kinetics; Enzyme Mechanisms; Enzymology; Hect Ligase; Humans; Hydrogen Bonding; Iodine Radioisotopes; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation; Phylogeny; Polyubiquitin - metabolism; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Sf9 Cells; Ubiquitin; Ubiquitin - metabolism; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Enzyme Mechanisms; Ubiquitin; E3 Ubiquitin Ligase; Hect Ligase; Enzyme Kinetics; Kinetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.517805

Employing 125I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508 Å2 and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe727 and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases kcat 62-fold but fails to affect E2∼ubiquitin binding or subsequent formation of the Hect domain Cys820∼ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe727-dependent trimer formation and catalytic activity (Ki = 12 mm), as does a conserved α-helical peptide corresponding to residues 474–490 of E6AP isoform 1 (Ki = 22 μm) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe727 intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (Kactivation = 1.5 nm) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the kcat defect of the Phe727 mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity. E6AP/UBE3A is a Hect ligase implicated in neurodevelopment and cell transformation. Kinetic/biophysical analyses demonstrate that E6AP oligomerization is required for polyubiquitin degradation signal assembly and that changes in oligomerization regulates such activity. E6AP oligomerization accounts for opposing effects of mutation and HPV16/18 E6 protein. These findings explain in part the etiology of specific Angelman syndrome mutations and E6-mediated cell transformation.