Figure 4. RING1 opening of E2~Ub enforces transfer via the RING2 active site.

1. Schematic representation of the two constructs used. Left: GST‐tagged HHARI_RBR with its native RING1 domain that does not induce closed conformations of UbcH5˜Ub. Right: GST‐tagged HHARI construct in which the RING1 domain was replaced with the Ubox domain of E4BU to generate the UBR hybrid that promotes closed conformations of UbcH5˜Ub.
2. Left: Auto‐ubiquitination assays were performed with wild‐type HHARI_RBR or an active‐site‐dead mutant (C357A‐HHARI_RBR). Products were visualized by Western blotting against GST. The zero time point was taken immediately prior to ATP addition; all other times are post‐ATP addition. Right: Identical assays as shown on left were performed with the UBR hybrid. The active‐site‐dead mutant (C357A) retains substantial auto‐ubiquitination activity signifying that UbcH5˜Ub is able to transfer Ub directly onto the GST‐UBR hybrid construct, bypassing the RING2 active site Cys.