Volume 58, Issue 2, Pages 297-310 (April 2015) Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin  Lori Buetow, Mads Gabrielsen, Nahoum G. Anthony, Hao Dou, Amrita Patel, Hazel Aitkenhead, Gary J. Sibbet, Brian O. Smith, Danny T. Huang  Molecular Cell  Volume 58, Issue 2, Pages 297-310 (April 2015) DOI: 10.1016/j.molcel.2015.02.017 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 58, 297-310DOI: (10.1016/j.molcel.2015.02.017) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 UbB Stimulates RING-Dependent and RING-Independent Ub Transfer (A) Reduced autoradiogram showing autoubiquitination by GST-RNF38-RING with 32P-Ub and UbcH5B variants over time. The asterisk (∗) represents non-reducible Ub-E1. (B) As in (A) but for full-length BIRC4 instead of GST-RNF38-RING. (C) Reduced autoradiogram showing ubiquitination of SMAC-32P by BIRC4 and UbcH5B variants over time. (D) Reduced autoradiograms showing diUb formation over time by UbcH5B variants with (top) or without RNF38-RING (bottom). Different exposure times were used for the panels. (E) Kinetics of diUb formation by RNF38-RING with varying concentrations of WT and UbcH5B S22R. Three rate replicates were measured for each UbcH5B concentration. Kinetic parameters and 95% CI are indicated. Error bars represent SD. (F) Non-reduced autoradiograms of pulsed-chased reactions showing the disappearance of UbcH5B∼32P-Ub variants over time with lysine in the presence and absence of K0UbΔGG or K0UbΔGG I44A, as indicated with RNF38-RING (top) or without E3 (bottom). (G) As for (F) but with UbcH5B∼32P-Ub, K0UbΔGG, the RING domains of RNF38 (top) and BIRC4 (middle) and UBE4B U-box (bottom). (H) Non-reduced antibiotin immunoblots of pulsed-chased lysine discharge reactions showing the disappearance of UbcH5B∼UbC over time with K0UbΔGG and RNF38-RING as indicated. (I) Non-reduced autoradiograms of pulsed-chased lysine discharge reactions showing the disappearance of UbcH5B∼32P-Ub variants over time with (left) or without RNF38-RING (right) in the presence and absence of K0UbΔGG. See also Figure S2. Molecular Cell 2015 58, 297-310DOI: (10.1016/j.molcel.2015.02.017) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 UbB Sources and Contributions to RING-Dependent and RING-Independent Ub Transfer (A) Non-reduced autoradiograms of pulsed-chased assays showing the simultaneous disappearance of UbcH5B∼32P-Ub variants and transfer of 32P-Ub to Ubn–GST-RNF38-RING over time. The asterisk (∗) is a mix of E1∼Ub and Ub-E1. (B) As in (A) but with Ubn–BIRC4. (C) Non-reduced autoradiograms showing the transfer of Ub to SMAC-32P over time by Ubn–BIRC4 in pulsed-chased reactions with indicated UbcH5B∼Ub variants. (D) As in Figure 1F but using K0UbΔGG or UbcH5BS22R F62A P95D–Ub and RNF38-RING. (E) Overlaid gel filtration Superdex 75 chromatograms of UbcH5B–Ub (red), UbcH5BS22R–Ub (blue), and gel filtration standards (GE Healthcare, orange and purple) with standard peaks and molecular weights indicated. See also Figure S3. Molecular Cell 2015 58, 297-310DOI: (10.1016/j.molcel.2015.02.017) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Ubn–BIRC4 and Ubn–SMAC Act as UbB Sources to Stimulate Ub Transfer (A) Non-reduced autoradiograms of pulsed-chased assays showing the simultaneous disappearance of UbcH5B∼32P-K0Ub variants and transfer of 32P-K0Ub to BIRC4. The asterisk (∗) is a mix of E1∼K0Ub and K0Ub-E1. (B) SDS-PAGE showing stopped BIRC4 and SMAC reactions used in (C). E1 was omitted from control reactions. (C) Non-reduced autoradiograms of pulsed-chased lysine discharge assays showing disappearance of UbcH5B∼32P-Ub variants over time by E3/substrate mixtures shown in (B). (D) As in (C) but mediated by BIRC4 mixed with indicated SMAC variants. Molecular Cell 2015 58, 297-310DOI: (10.1016/j.molcel.2015.02.017) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Structures of E3-E2–Ub and E3-UbB-E2–Ub (A) Cartoon representation of the E3-E2–Ub complex. Left and right are related by 90° rotation about the y axis. UbcH5B is colored cyan, UbD yellow, and RNF38-RING gray. The UbcH5B–Ub linkage is indicated with an arrow and UbcH5B’s structural components labeled. (B) Cartoon representation of the E3-UbB-E2–Ub. UbB is colored orange and all other features are colored or highlighted as in (A). (C) Ribbon diagram showing superposition of E3-UbB-E2–Ub colored black with UbB omitted and both copies of E3-E2–Ub from the asymmetric unit colored red and wheat. The UbcH5B α1β1 loop is indicated by a black arrow. Left and right are related by 90° rotation about the y axis and the view is identical to those shown in (A) and (B). See also Figures S4 and S5. Molecular Cell 2015 58, 297-310DOI: (10.1016/j.molcel.2015.02.017) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Mechanistic Aspects of UbB-Stimulation Based on Structural Comparison of E3-E2–Ub and E3-UbB-E2–Ub (A) Close-up of UbB-UbcH5B interaction in E3-UbB-E2–Ub. The color scheme is as described in Figure 4A with the side chains of key residues shown as sticks. O atoms are red. S atoms are orange, and N atoms are blue. UbcH5B components that contact UbB are labeled. (B) Close-up of UbD-UbcH5B α1β1 loop-UbB interface with interacting residues shown as sticks and colored as described in (A). Putative hydrogen bonds are shown as dashed lines. (C) Non-reduced autoradiograms of pulsed-chased reactions as in Figure 1I. (D) RNF38-RING mediated hydrolysis of UbcH5BC85S–Ub variants over time in the presence and absence of K0UbΔGG visualized by SDS-PAGE. (E) Close-up of UbD-UbcH5B α1β1 loop-UbB from superposition of E3-E2–Ub and E3-UbB-E2–Ub. Colored as described in (B). (F) Non-reduced autoradiograms of pulsed-chased reactions as in C but with UbcH5B∼32P-Ub and K0UbΔGG variants. See also Figure S6. Molecular Cell 2015 58, 297-310DOI: (10.1016/j.molcel.2015.02.017) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Mechanism of UbB-Stimulated E3-Mediated Ub Transfer (A) UbB-stimulated E3-mediated Ub transfer proceeds via a sequential mechanism where E3 initially recruits UbcH5B∼Ub and enhances UbB-UbcH5B∼Ub binding affinity. E3-E2∼Ub is allosterically activated by UbB, which secures UbcH5B’s α1 and α1β1 loop into a conformation that supports E3-mediated Ub transfer. UbB-UbcH5B∼Ub binding affinity is enhanced to an extent where estimated cellular concentrations of free Ub can potentially saturate UbcH5B’s backside for stimulated Ub transfer. Conjugated Ub from Ubn–E3 and Ubn–substrate can act as UbB sources in cis to amplify UbB-stimulated Ub transfer. (B) Thermodynamic model of E3-UbB synergistic binding enhancement. The formation of E3-UbB-E2–Ub complex can be dissected into a four-step thermodynamic cycle based on free energies (ΔG) calculated from experimental dissociation constants (Table 1). (i) Dissociation of UbB-UbcH5B–Ub to UbB and UbcH5B–Ub. (ii) Dissociation of RNF38-RING-UbB-UbcH5B–Ub to RNF38-RING and UbB-UbcH5B–Ub. (iii) Dissociation of RNF38-RING-UbB-UbcH5B–Ub to RNF38-RING-UbcH5B–Ub and UbB. (iv) Dissociation of RNF38-RING-UbcH5B–Ub to RNF38-RING and UbcH5B–Ub. Molecular Cell 2015 58, 297-310DOI: (10.1016/j.molcel.2015.02.017) Copyright © 2015 Elsevier Inc. Terms and Conditions