Evidence for Bidentate Substrate Binding as the Basis for the K48 Linkage Specificity of Otubain 1

Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear α-linked polyubiqui...

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Bibliographic Details
Published in:	Journal of molecular biology 2009-03, Vol.386 (4), p.1011-1023
Main Authors:	Wang, Tao, Yin, Luming, Cooper, Eric M., Lai, Ming-Yih, Dickey, Seth, Pickart, Cecile M., Fushman, David, Wilkinson, Keith D., Cohen, Robert E., Wolberger, Cynthia
Format:	Article
Language:	eng
Subjects:	Affinity Labels Animals Binding Sites Caenorhabditis elegans Cysteine Endopeptidases - chemistry Cysteine Endopeptidases - metabolism Deubiquitinating Enzymes deubiquitination Humans isopeptide linkage specificity Lysine - metabolism Magnetic Resonance Spectroscopy Models, Molecular otubain Peptide Fragments - metabolism polyubiquitin Polyubiquitin - metabolism Protein Binding Protein Structure, Tertiary Substrate Specificity Sulfones
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Summary:	Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear α-linked polyubiquitin. Cleavage is not limited to either end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Intriguingly, cleavage of K48-diubiquitin by hOtu1 can be inhibited by diubiquitins of various linkage types, as well as by monoubiquitin. NMR studies and activity assays suggest that both the proximal and distal units of K48-diubiquitin bind to hOtu1. Reaction of Cys23 with ubiquitin-vinylsulfone identified a ubiquitin binding site that is distinct from the active site, which includes Cys91. Occupancy of the active site is needed to enable tight binding to the second site. We propose that distinct binding sites for the ubiquitins on either side of the scissile bond allow hOtu1 to discriminate among different isopeptide linkages in polyubiquitin substrates. Bidentate binding may be a general strategy used to achieve linkage-specific deubiquitination.
ISSN:	0022-2836 1089-8638