Specificity of the BRISC Deubiquitinating Enzyme Is Not Due to Selective Binding to Lys63-linked Polyubiquitin

• Published in: The Journal of biological chemistry Vol. 285; no. 14; pp. 10344 - 10352
• Main Authors: Cooper, Eric M., Boeke, Jef D., Cohen, Robert E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.04.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Binding Sites; Carbon-Nitrogen Lyases - metabolism; Catalytic Domain; Deubiquitinating Enzymes; Enzymes/Kinetics; Enzymes/Metallo; Enzymes/Proteolytic; HeLa Cells; Humans; Lysine - metabolism; Membrane Proteins - metabolism; Multiprotein Complexes - genetics; Multiprotein Complexes - metabolism; Polyubiquitin - metabolism; Proteases/Metalloprotease; Proteases/Ubiquitin; Proteases/Ubiquitination; Proteasome Endopeptidase Complex - metabolism; Protein Binding; Protein Synthesis and Degradation; Protein/Post-translational Modification; Protein/Turnover; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity; Trans-Activators - metabolism; Ubiquitination; Ubiquitins - metabolism; Protein/Turnover; Protein/Post-translational Modification; Enzymes/Kinetics; Enzymes/Proteolytic; Proteases/Ubiquitin; Proteases/Metalloprotease; Proteases/Ubiquitination; Enzymes/Metallo
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.059667

BRISC (Brcc36-containing isopeptidase complex) is a four-subunit deubiquitinating (DUB) enzyme that has a catalytic subunit, called Brcc36, that is a member of the JAMM/MPN+ family of zinc metalloproteases. A notable feature of BRISC is its high specificity for cleaving Lys63-linked polyubiquitin. Here, we show that BRISC selectivity is not due to preferential binding to Lys63-linked polyubiquitin but is instead dictated by how the substrate isopeptide linkage is oriented within the enzyme active site. BRISC possesses a high affinity binding site for the ubiquitin hydrophobic surface patch that accounts for the bulk of the affinity between enzyme and substrate. Although BRISC can interact with either subunit of a diubiquitin conjugate, substrate cleavage occurs only when BRISC is bound to the hydrophobic patch of the distal (i.e. the “S1”) ubiquitin at a ubiquitin-ubiquitin cleavage site. The importance of the Lys63-linked proximal (S1′) ubiquitin was underscored by our finding that BRISC could not cleave the isopeptide bond joining a ubiquitin to a non-ubiquitin substrate. Finally, we also show that Abro1, another BRISC subunit, binds directly to Brcc36 and that the Brcc36-Abro1 heterodimer includes a minimal complex with Lys63-specific DUB activity.