Specificity of the BRISC deubiquitinating enzyme is not due to selective binding to Lys63-linked polyubiquitin.
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 Lys(63)-linked polyubiquitin. Here, we show that BRISC selectivity is not due to preferential binding to Lys(63)-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 Lys(63)-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 Lys(63)-specific DUB activity.
Cooper
Eric M
EM
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205, USA. emcooper@jhsph.edu
Boeke
Jef D
JD
Cohen
Robert E
RE
D001665:Binding Sites; D019759:Carbon-Nitrogen Lyases / Q000378:metabolism; D020134:Catalytic Domain; D000072017:Deubiquitinating Enzymes; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D025821:Polyubiquitin / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism*
D008565:Membrane Proteins; D046912:Multiprotein Complexes; C109285:PSMD14 protein, human; D011994:Recombinant Proteins; D015534:Trans-Activators; D014452:Ubiquitins; C075986:diubiquitin conjugate; D025821:Polyubiquitin; C518017:BRCC3 protein, human; D000072017:Deubiquitinating Enzymes; D046988:Proteasome Endopeptidase Complex; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; D008239:Lysine