title: Association of the disordered C-terminus of CDC34 with a catalytically bound ubiquitin.
abstract: Cell division cycle protein 34 (CDC34) is a key E2 ubiquitin (Ub)-conjugating enzyme responsible for the polyubiquitination of proteins controlling the G1/S stages of cell division. The acidic C-terminus of the enzyme is required for this function, although there is little structural information providing details for a mechanism. One logical time point involving the C-terminus is the CDC34-Ub thiolester complex that precedes Ub transfer to a substrate. To examine this, we used a CDC34-Ub disulfide complex that structurally mimics the thiolester intermediate. NMR spectroscopy was used to show that the CDC34 C-terminus is disordered but can intramolecularly interact with the catalytically bound Ub. Using chemical shift perturbation analysis, we mapped two interacting regions on the surface of Ub in the CDC34-Ub complex. The first site comprises a hydrophobic patch (typical of other Ub complexes) that associates with the CDC34 catalytic domain. A novel second site, dependent on the C-terminus of CDC34, comprises a lysine-rich surface (K6, K11, K29, and K33) on the opposite face of Ub. Further, NMR experiments show that this interaction is described by two slowly exchanging states-a compact conformation where the C-terminus of CDC34 interacts with bound Ub and an extended structure where the C-terminus is released. This work provides the first structural details that show how the C-terminus of CDC34 might direct a thiolester-bound Ub to control polyubiquitin chain formation.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
authors.forename: Donald E
authors.initials: DE
authors.lastname: Spratt
authors.forename: Gary S
authors.initials: GS
authors.lastname: Shaw
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D001665:Binding Sites; D020134:Catalytic Domain; D002453:Cell Cycle; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D025821:Polyubiquitin / Q000378:metabolism; D011487:Protein Conformation; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D025801:Ubiquitin; D025821:Polyubiquitin; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D008239:Lysine