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Functional characterization of UbcH7 interaction with the E6AP ubiquitin ligase
Author(s) -
Ronchi Virginia Paola,
Haas Arthur L
Publication year - 2009
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.23.1_supplement.848.7
Subject(s) - ubiquitin , ubiquitin ligase , chemistry , elongation , biophysics , biochemistry , stereochemistry , microbiology and biotechnology , kinetics , binding site , biology , materials science , physics , gene , quantum mechanics , ultimate tensile strength , metallurgy
E6AP is the founding member of the HECT domain superfamily of ubiquitin ligases, mutations in which are responsible for Angelman syndrome. Kinetic analysis of polyubiquitin chain formation shows UbcH7 is the functionally cognate E2 for E6AP, the concentration dependence of which yields a K m =58±6 nM and k cat =0.013 s −1 . Other kinetic studies indicate E6AP harbors two UbcH7 binding sites: Site 1 involved in the initial formation of the Hect domain~Ub thiolester and Site 2 required for chain elongation. Mutations designed to disrupt Site 2 interactions predicted from the crystal structure support the model. UbcH7K100A shows nearly indistinguishable kinetics with K m = 31 ± 17 nM and k cat =0.081±0.008 s −1 , indicating Site 1 using binding interactions distinct from Site 2. UbcH7F63A forms the initial Hect~Ub thiolester but doe not support chain elongation even though it binds E6AP and is a competitive inhibitor of Site 1 bound UbcH7 (K i =410 nM). Pulse‐chase kinetics with 125 I‐ubiquitin confirms chain elongation occurs by distal addition to the growing polyubiquitin chain. RAD23A, substrate of E6AP, negatively affects the polyubiquitination of substrates by Ubc2. The kinetic analysis shows that RAD23A was an uncompetitive inhibitor of the conjugation reaction catalyzed by UbcH7‐E6AP ( K i 414 nM). These studies reveal the complex mechanism of interaction among UbcH7, E6AP and RAD23A. (Supported by GM340092 to A.L.H.).