Fig. 3. UBE2K acceptor site employs UBC and UBA to orient Ub^A.

a, Ub^A (magenta) is encased by UBE2K UBC (cyan) and UBA (blue). b, Close-up views of key residues (sticks) between Ub^A and UBC domain in a. Hydrogen bonds are shown as dotted lines. Asterisks (*) indicate modeled residues. c, Interaction between Ub^A and UBA domain with key residues shown as sticks. d, Nonreduced SDS–PAGE showing UBE2K-catalyzed di-Ub formation with Ub^A variants. I36, R42 and I44 are remote from the UBE2K/Ub^A binding interface. e, Nonreduced SDS–PAGE showing di-Ub formation using UBE2K with acceptor-site mutations. Asterisks (*) in d and e indicate fluorescent-labeled Ub^D. f, Kinetics of di-Ub formation catalyzed by UBE2K variants. Data from two independent experiments (n = 2) were fitted with the Michaelis–Menten equation. k_cat/K_m values (UBE2K^WT = 750 M⁻¹ min⁻¹, UBE2K^M172D,L198R = 684 M⁻¹ min⁻¹ and UBE2K^L186A = 355 M⁻¹ min⁻¹) were estimated from the slope of the linear portion of curve.

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