Figure 6.

Ub^B interactions. A, cartoon representation showing the UbcH5B–Ub^B portion of the structure from the cIAP1R-UbcH5B–Ub-Ub^B complex. Ile⁴⁴ of Ub^B and Ser²² of UbcH5B are indicated. B, close-up view of Ub^B-UbcH5B–Ub^D binding interface. UbcH5B's α1β1-loop is indicated by an arrow. Hydrogen bonds are shown as dotted lines. All coloring in A and B is the same as in Fig. 2. C, nonreduced SDS-PAGE of lysine discharge reactions showing the disappearance of UbcH5B variant∼Ub bands over time in the presence and absence of UbΔGG catalyzed by cIAP1R. *, contaminating band from other reaction components. D, close-up view of Ub^B-UbcH5B–Ub^D binding interface in the structure of RNF38-UbcH5B–Ub-Ub^B complex (PDB entry 4V3L). UbcH5B is shown in cyan, Ub^D in yellow, and Ub^B in wheat. E, close-up view of UbcH5B's α1β1-loop in the structure of RNF38-UbcH5B–Ub complex (PDB entry 4V3K). UbcH5B is shown in cyan and Ub^D in yellow. D and E are shown in the same orientation as in B. F, comparison of Ub^B conformations in the structures of cIAP1R-UbcH5B–Ub-Ub^B and RNF38-UbcH5B–Ub-Ub^B complexes (PDB entry 4V3L). Superimposition was performed on all Cα atoms of the UbcH5B portion of the structure. Ribbon representations of the UbcH5B-Ub^B portion from both structures are shown. Ub^B's β1β2 loop is indicated by an arrow. UbcH5B and Ub^B from cIAP1R-UbcH5B–Ub-Ub^B structure are colored as in Fig. 2. UbcH5B and Ub^B from RNF38-UbcH5B–Ub-Ub^B structure are colored in cyan and gray, respectively. G, nonreduced SDS-PAGE of lysine discharge reactions showing the disappearance of the UbcH5B∼Ub band over time in the presence of excess WT Ub or Ub K6A catalyzed by cIAP1R.