Fig. 2. Validation of the UBE2K/Ub^D/RING network.

a, Structure of Ub^D (yellow) with the C terminus covalently attached to the UBE2K (cyan) active site K92 and primed for catalysis by RNF38 RING (gray). The isopeptide bond between UBE2K K92 and Ub^D G76 along with key residues in the binding interface are shown as sticks. b, Nonreduced SDS–PAGE showing di-Ub formation with the RING-binding-deficient UBE2K mutant. Asterisks (*) indicate fluorescent-labeled Ub. c, Nonreduced SDS–PAGE showing di-Ub formation in the presence of RNF38 E2/Ub^D-binding-deficient mutants. d, Nonreduced SDS–PAGE showing the effect of Ub^D mutations on di-Ub formation with RNF38. e, Nonreduced SDS–PAGE of RNF38 with UBE2K^D94A and UBE2K^A115R in di-Ub formation. Coomassie staining was used to visualize c, d and e.

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