title: Crystal structure of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Rad6 at 2.6 A resolution.
abstract: The Saccharomyces cerevisiae ubiquitin-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 A resolution to an R factor of 21.3%. The protein adopts an alpha/beta 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.
authors.affiliation: Biochemistry Department, University of Utah, Salt Lake City, Utah 84132, USA.
authors.forename: D K
authors.initials: DK
authors.lastname: Worthylake
authors.forename: S
authors.initials: S
authors.lastname: Prakash
authors.forename: L
authors.initials: L
authors.lastname: Prakash
authors.forename: C P
authors.initials: CP
authors.lastname: Hill
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013499:Surface Properties; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C063607:RAD18 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; C066066:UBR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D008025:Ligases