Structural Basis for Ubiquitin Recognition by the Otu1 Ovarian Tumor Domain Protein

Troy Eugene Messick,Nathaniel Scott Russell,Ayaka Jennifer Iwata,Kathryn Lorenz Sarachan,Ramin Shiekhattar,John R Shanks,Francisca E Reyes-Turcu,Keith D Wilkinson,Ronen Marmorstein

doi:10.1074/jbc.m704398200

Troy Eugene Messick, Nathaniel Scott Russell + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m704398200

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Abstract

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of approximately 130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys(48) linkages, having little or no activity on Lys(63)- and Lys(29)-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

Highlights

Deubiquitination involves the hydrolysis of these isopeptide bonds and requires the activity of a deubiquitinating enzyme (DUB) [8]
DUBs can be divided into six distinct families: the ubiquitin-specific processing proteases (UBPs), the ubiquitin carboxyl-terminal hydrolases (UCHs), the Ataxin-3/Josephin domains, the ovarian tumor domain-containing proteases (OTUs) [9, 10], the viral processing proteases [11, 12], and the JAMM proteases
The JAMM metalloproteases are unique among the DUBs in that zinc is required to catalyze the reaction [13], whereas the other DUB families use an active site cysteine to hydrolyze the isopeptide bond between ubiquitin and the target

Summary

EXPERIMENTAL PROCEDURES

Preparation of the Otu1-Ubiquitin Complex—The genes encoding amino acids 87–301 of Otu and full-length ubiquitin were PCR-amplified and cloned into the pTYB2 vector. To create the Otu1-ubiquitin complex, concentrated Otu was mixed with Ub-Br3 in 1:1 stoichiometric amounts in a solution containing 100 mM HEPES, pH 7.4, and incubated for 2 h at 37 °C. The complex was enriched using a HiTrap Q column (Amersham Biosciences) with a NaCl gradient at pH 8.5 and further purified on a Superdex-200 gel filtration column equilibrated with phosphate-buffered saline, pH 7.4, and 1 mM Tris(2-carboxyethyl)phosphine. Orthorhombic crystals were obtained by mixing 2 ␮l of 15 mg/ml protein complex with 2 ␮l of reservoir solution and equilibrating over 0.5 ml of reservoir solution containing 100 mM Bis-Tris, pH 6.5, 100 –250 mM magnesium chloride, 16 –21% polyethylene glycol 3350. A hexagonal crystal form of the protein complex was prepared by mixing 2 ␮l of 15 mg/ml protein complex with 2 ␮l of reservoir and equilibration solution containing 100 mM MES, pH 6.5, 50 –200 mM ammonium acetate, 17–22% polyethylene glycol 3350. Resolution (Å) 50-1.50 (1.55-1.50) 20-2.0 (2.07-2.00) 20-2.0 (2.07-2.00) 20-2.0 (2.07-2.00) 50-2.31 (2.39-2.31) 50-2.31 (2.39-2.31) 50-2.45 (2.56-2.45)

Root mean square

Is oT control

Ruby Red staining

Findings

Ub Contacts