Dataset Information

Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein.

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.

SUBMITTER: Messick TE

PROVIDER: S-EPMC2447653 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein.

Messick Troy Eugene TE Russell Nathaniel Scott NS Iwata Ayaka Jennifer AJ Sarachan Kathryn Lorenz KL Shiekhattar Ramin R Shanks John R JR Reyes-Turcu Francisca E FE Wilkinson Keith D KD Marmorstein Ronen R

The Journal of biological chemistry 20080212 16

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. ...[more]

PMID: 18270205

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Project description:Pathogenic bacteria have acquired a vast array of eukaryotic-protein-like proteins via intimate interaction with host cells. Bacterial effector proteins that function as ubiquitin ligases and deubiquitinases (DUBs) are remarkable examples of such molecular mimicry. LotA, a Legionella pneumophila effector, belongs to the ovarian tumor (OTU) superfamily, which regulates diverse ubiquitin signals by their DUB activities. LotA harbors two OTU domains that have distinct reactivities; the first one is responsible for the cleavage of the K6-linked ubiquitin chain, and the second one shows an uncommon preference for long chains of ubiquitin. Here, we report the crystal structure of a middle domain of LotA (LotAM), which contains the second OTU domain. LotAM consists of two distinct subdomains, a catalytic domain having high structural similarity with human OTU DUBs and an extended helical lobe (EHL) domain, which is characteristically conserved only in Legionella OTU DUBs. The docking simulation of LotAM with ubiquitin suggested that hydrophobic and electrostatic interactions between the EHL of LotAM and the C-terminal region of ubiquitin are crucial for the binding of ubiquitin to LotAM. The structure-based mutagenesis demonstrated that the acidic residue in the characteristic short helical segment termed the "helical arm" is essential for the enzymatic activity of LotAM. The EHL domain of the three Legionella OTU DUBs, LotA, LotB, and LotC, share the "helical arm" structure, suggesting that the EHL domain defines the Lot-OTUs as a unique class of DUBs. IMPORTANCE To successfully colonize, some pathogenic bacteria hijack the host ubiquitin system. Legionella OTU-like-DUBs (Lot-DUBs) are novel bacterial deubiquitinases found in effector proteins of L. pneumophila. LotA is a member of Lot-DUBs and has two OTU domains (OTU1 and OTU2). We determined the structure of a middle fragment of LotA (LotAM), which includes OTU2. LotAM consists of the conserved catalytic domain and the Legionella OTUs-specific EHL domain. The docking simulation with ubiquitin and the mutational analysis suggested that the acidic surface in the EHL is essential for enzymatic activity. The structure of the EHL differs from those of other Lot-DUBs, suggesting that the variation of the EHL is related to the variable cleaving specificity of each DUB.

Dataset Information

Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein.

Publications

Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein.

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