FIGURE 7.

UbcH5 binds preferentially to monoubiquitinated Otub1 through backside UbcH5-Ub interaction. A, UbcH5 binds preferentially to the monoubiquitinated Otub1. His-Otub1 was subjected to an in vitro ubiquitination reaction as in Fig. 1. The reaction mixture containing both monoubiquitinated and unmodified Otub1 was then incubated with GST alone or GST-UbcH5c immobilized onto GSH beads. After washing, bead-bound proteins were assayed by IB analysis. B, modeling of the Ub-Otub1/UbcH5b interaction through docking of Ub linked to either Lys-59 (top left panel) or Lys-109 (bottom left panel) with the backside of UbcH5b. Otub1, UbcH5b, and Ub are colored green, blue, and yellow, respectively. Lys-59 and Lys-109 of Otub1, Ile-44 of Ub, and Ser-22 of UbcH5 are indicated in red. The enlarged view in the right panel shows the canonical Ub Ile-44 interacting with Ser-22 on UbcH5. C and D, UbcH5^S22R and UbcH5^S22L do not bind to monoubiquitinated Otub1. The in vitro ubiquitination reaction mixture containing both monoubiquitinated and unmodified Otub1 was incubated with GST alone, GST-UbcH5c, GST-UbcH5^S22R, or GST-UbcH5^S22L immobilized onto GSH beads. After washing, bead-bound proteins were assayed by IB analysis. E, mutating Ser-22 to Leu rescues the binding defect of UbcH5 with Otub1-Ub^I44A. His-Otub1 was subjected to an in vitro ubiquitination reaction using recombinant Ub^I44A. The reaction mixture containing both monoubiquitinated (Otub1-Ub^I44A) and unmodified Otub1 was incubated with GST alone, GST-UbcH5c, or GST-UbcH5^S22L immobilized onto GSH beads. After washing, bead-bound proteins were assayed by IB analysis.