PROTEIN CHEMISTRY AND STRUCTURE
Crystal Structure of the Saccharomyces cerevisiae Ubiquitin-conjugating Enzyme Rad6 at 2.6 Å Resolution*

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The Saccharomyces cerevisiaeubiquitin-conjugating enzyme (UBC) Rad6 is required for several functions, including the repair of UV damaged DNA, damage-induced mutagenesis, sporulation, and the degradation of cellular proteins that possess destabilizing N-terminal residues. Rad6 mediates its role in N-end rule-dependent protein degradation via interaction with the ubiquitin-protein ligase Ubr1 and in DNA repair via interactions with the DNA binding protein Rad18. We report here the crystal structure of Rad6 refined at 2.6 Å resolution to an R factor of 21.3%. The protein adopts an α/β fold that is very similar to other UBC structures. An apparent difference at the functionally important first helix, however, has prompted a reassessment of previously reported structures. The active site cysteine lies in a cleft formed by a coil region that includes the 310 helix and a loop that is in different conformations for the three molecules in the asymmetric unit. Residues important for Rad6 interaction with Ubr1 and Rad18 are on the opposite side of the structure from the active site, indicating that this part of the UBC surface participates in protein-protein interactions that define Rad6 substrate specificity.

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This work was supported by National Institutes of Health Grants GM50163 and GM19261 and American Cancer Society Grant JFRA B-74386.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1ayz and r1ayzsf) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

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Supported by National Institutes of Health predoctoral training Grant 5-T32-GM08573.