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. 2019 Oct 22;8:e49223. doi: 10.7554/eLife.49223

Figure 10. Activation of UBC13 ~ Ub conjugates by RING domains.

(A) A comparison of Ub conjugation to UBC13 and dUBC13. E1, ATP, and UBC13/dUBC13 were incubated in reaction buffer for 15 min, quenched by adding EDTA and separated on SDS page. The amount of E2 ~ Ub conjugates were quantified and plotted in the right section. The values are the mean of three reactions, and the error is the standard deviation of the same. (B) Affinity pull-down experiment was performed by incubating MBP beads with MBP-Mms2, UBC13 ~ Ub (or dUBC13 ~ Ub) and TRAF6RZ3, washed thoroughly and separated on SDS gels. The asterisk denotes impurities. (C) Affinity pull-down experiment was performed by incubating GST beads with GST-RNF38RING and UBC13 ~ Ub (or dUBC13 ~ Ub), washed thoroughly and separated on SDS gels. (D) Single-round discharge of Ub from UBC13 ~ Ub and dUBC13 ~ Ub catalyzed by TRAF6RING was monitored. UBC13 was conjugated with Ub, and the reaction was quenched. Then Mms2, TRAF6RING, and Lysine were added to the reaction mixture, and E2 ~ Ub and free E2 was monitored over time. The proteins bands in (D) were quantified and plotted in (G) and (H). The plotted values are the mean of triplicates, and the error is the standard deviation of the same. (E) Same as in D), where the discharge is catalyzed by RNF38RING domain. The proteins in (E) are quantified and plotted in (I) and (J). The rate of discharge in each case was calculated as discharge-rate = (Total E2-E2 ~ Ub)/(Total E2.time) for the initial time points and given in (F). (K) The rate of Ub2 synthesis was monitored over time. UBC13 or dUBC13 was conjugated with Ub, and the reaction was quenched. Then Mms2, RNF38RING and Acceptor Ub was added to the reaction mixture, and the synthesis of Ub2 was monitored over time. (L) The rate of Ub2 synthesis for UBC13 and dUBC13 are compared at substrate (acceptor-Ub) concentration of 50 μM. (M) The reaction rates for UBC13 and dUBC13 are compared at various substrate concentrations. (N) The kinetic parameters of Ub2 synthesis for UBC13 and dUBC13 are given in a table. The values are the mean of triplicates, and the error is the standard deviation of the same. The Ub used in all the experiments of this figure is K63A-Ub, except the acceptor Ub used in (K)-(M) is D77-Ub.

Figure 10.

Figure 10—figure supplement 1. Kinetics of Ub conjugation, RING-binding, and Ub-discharge.

Figure 10—figure supplement 1.

(A) The complete gel-image of the Ub-conjugation reaction shown in Figure 10A. (B) The complete gel-image of TRAF6-RZ3 and UBC13 ~ Ub binding experiment of Figure 10B. (C) The control UBC13 ~ Ub discharge experiment in the absence of the RING domain. (D) The dUBC13 ~ Ub discharge experiment with RNF38(5X).
Figure 10—figure supplement 2. Kinetics of Ub2 synthesis.

Figure 10—figure supplement 2.

(A) The kinetics of Ub2 synthesis in UBC13/Mms2 the absence of RING domain. The control lane C is purified Ub2. (B) The kinetics of Ub2 synthesis in dUBC13/Mms2 the absence of RING domain. The amount of Ub2 formed by (C) UBC13/Mms2/RNF38RING and (D) dUBC13/Mms2/RNF38RING is plotted against time. The fits of the data are shown as dotted lines. The various substrate (D77-Ub) concentrations are shown by different colors.
Figure 10—figure supplement 3. Stabilization of the UBC13 ~ Ub conjugate by the TRAF6 homodimer.

Figure 10—figure supplement 3.

Structure of the UBC13 ~ Ub-TRAF6RING catalytic complex (PDB 5VO0). The inset shows two essential Arginines located on the distal TRAF6 protomer (dTRAF6), which help to stabilize the closed conformation of UBC13 ~ Ub in addition to the proximal unit (pTRAF6).