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J. Biol. Chem., Vol. 280, Issue 32, 29176-29185, August 12, 2005

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Structural Basis for Interaction between the Ubp3 Deubiquitinating Enzyme and Its Bre5 Cofactor^*

Keqin Li, Kehao Zhao, Batool Ossareh-Nazari, Guoping Da¶, Catherine Dargemont, and Ronen Marmorstein¶||

From the The Wistar Institute and the ^¶Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the Institute Jacques Monod, UMR7592 CNRS/Paris VI/Paris VII, Paris 75251, France

The Bre5 protein is a cofactor for the deubiquitinating enzyme Ubp3, and it contains a nuclear transfer factor 2 (NTF2)-like protein recognition module that is essential for Ubp3 activity. In this study, we report the x-ray crystal structure of the Bre5 NTF2-like domain and show that it forms a homodimeric structure that is similar to other NTF2-like domains, except for the presence of an intermolecular disulfide bond in the crystals. Sedimentation equilibrium studies reveal that under non-reducing conditions, the Bre5 NTF2-like domain is exclusively dimeric, whereas a disulfide bond-deficient mutant undergoes a monomer-dimer equilibrium with a dissociation constant in the midnanomolar range, suggesting that dimer formation and possibly also disulfide bond formation may modulate Bre5 function in vivo. Using deletion analysis, we also identify a novel N-terminal domain of Ubp3 that is necessary and sufficient for interaction with Bre5 and use isothermal titration calorimetry to show that Bre5 and Ubp3 form a 2:1 complex, in contrast to other reported NTF2-like domain/protein interactions that form 1:1 complexes. Finally, we employ structure-based mutagenesis to map the Ubp3 binding surface of Bre5 to a region near the Bre5 dimer interface and show that this binding surface of Bre5 is important for Ubp3 function in vivo. Together, these studies provide novel insights into protein recognition by NTF2-like domains and provide a molecular scaffold for understanding how Ubp3 function is regulated by Bre5 cofactor binding.

Received for publication, March 17, 2005 , and in revised form, June 7, 2005.

The atomic coordinates and structure factors (code 1ZX2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from the Minister for Research and the Association de Recherche contre le Cancer (to C. D.) and from the National Institutes of Health (GM60293) (to R. M.) and by a grant from the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health (to the Wistar Institute). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: The Wistar Institute, Philadelphia, PA 19104. Tel.: 215-898-5006; Fax: 215-898-0381; E-mail: marmor{at}wistar.org.

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