Association of the disordered C-terminus of CDC34 with a catalytically bound ubiquitin

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.