Fig. 3.
The A96D-UbcH5 ∼ Ub conjugate is impaired in reactivity with SspH2. (A) Ribbon diagram of UbcH5 highlighting the SspH2 contact region (Red) and locations of mutations described in the text. Ala 96 (Red Spheres) resides within the mapped contact region whereas R5, S22, F62, and K63 (Gray Spheres) lie outside this region. The UbcH5 active site residue C85 is shown in yellow. (B) (A96D)-UbcH5 ∼ Ub exhibits reduced activity with SspH2 in the synthesis of poly-Ub chains relative to wt-UbcH5, whereas (F62A)-UbcH5 ∼ Ub, like R5A, S22R, and K63A (14, 15), is only moderately affected. Reactions were conducted with 10 μM E2 ∼ UbT7 and 1 μM SspH2477–788 at 32 °C. Samples were collected at 0, 10, and 20 minutes after addition of 5 mM ATP and visualized by western analysis with α-T7 antibodies.