Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases

Abstract

Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here, we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 side chain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates.

Figure 1. Model of the E4BU:UbcH5c~Ub complex

a) The E4BU:UbcH5 complex (PDB ID 3L1Z) is shown as a representative structure of RING/U-box E3:E2 structures. Structural features relevant to this work are noted. b) A side-on view of the UbcH5c~Ub structural ensemble generated from small angle X-ray data (Pruneda et al., 2011). c) Five representative structures of the top-scored HADDOCK cluster, aligned on UbcH5c (green); E4BU and Ub are shown in blue and red, respectively. For comparison, the view on the left is comparable to that shown in panel b). d) An electrostatic surface is shown for a representative E4BU:UbcH5c~Ub structure within the top-scored HADDOCK cluster. Note the expansive interaction between an acidic patch on UbcH5c and a basic region on Ub. e) Regions affected by the spin-labeled conjugate (i.e., Ub-K11SL) are painted in magenta (> 1.5 sd from the mean I_act/I_red); the alpha carbon of Ub residue 11 from one member of the ensemble is shown as a yellow sphere for reference. f) Mesh representation of the axially symmetric rotational diffusion tensors for Ub from the UbcH5c-O~Ub conjugate (left, green) and the ternary complex with E4BU (middle, blue), and aligned and superimposed (right).

Figure 2. Mutations that disable RING/U-box E3-mediated UbcH5c~Ub conformational change disrupt activity

a) Close-up of protein-protein interactions within the ternary complex model. Residues chosen for mutation (yellow sticks) include E4BU F1141 and R1143, UbcH5c L104, and Ub L8, I44, V70, and T12. E4BU L1107 (mutation to Ala disrupts E3:E2 interaction) is shown for reference. b) Auto-ubiquitination activity assays with BC₃₀₄/BD₃₂₇ and UbcH5c, as monitored by Western analysis following FLAG-BRCA1. Reaction components are WT except when indicated. c) Lysine reactivity assays with BC₁₁₂/BD₁₄₀ and UbcH5c. Reaction components are WT except when indicated. Western blot for HA-Ub(K0) to monitor decay of the UbcH5c~HA-Ub(K0) conjugate is shown. Two reaction time courses are shown for each E2~Ub: on the left, time course in the presence of WT BRCA1/BARD1 (specify the construct) and on the right, time course in the presence of I26A-BRCA1/BARD1, which does not bind to UbcH5 (Brzovic et al., 2003). d) Time course of the hydrolysis of UbcH5c oxyesters in the presence and absence of E4BU, as followed by 1-D NMR. The y-axis shows the fraction of oxy-ester remaining, as measured by integration of the UbcH5c Asn77 resonance at its conjugated position. Curves for UbcH5c-O~Ub and E4BU:UbcH5c-O~Ub samples are presented in black and yellow, respectively. Replacement of WT components in the E4BU:UbcH5c-O~Ub complex with Ub^I44A, UbcH5c^L104Q, or E4BU^R1143A are colored in red, green, and blue, respectively.

Figure 3. Conformational activation of the E2~Ub conjugate is required for RING/U-box E3 activity

a) Lysine reactivity assays for Ube2e1, Ube2n, and Ube2k with BC₁₁₂/BD₁₄₀. E2 identity is listed in column at left. Western blot for HA-Ub(K0) to follow decay of the E2~HA-Ub(K0) conjugate is shown. b) Auto-ubiquitination assays of RING/U-box E3s with UbcH5c^WT and UbcH5c^L104Q. Western blot following HA-Ub is shown. c) As in b), but with HECT-type E3s, as indicated.

Figure 4. A conserved E3:E2 hydrogen bond is required for E3-mediated Ub transfer

a) Examples of RING/U-box E3:E2 interactions observed in deposited crystal structures include E4B:UbcH5c (3L1Z, left), TRAF6:Ubc13 (3HCT, center), and c-Cbl:UbcH7 (1FBV, right). 3L1Z represents the common case in which a conserved E3 basic residue directly hydrogen bonds the E2 backbone. TRAF6, which lacks the conserved basic residue, maintains this interaction through an ordered water molecule. In contrast, no hydrogen bond is observed in the c-Cbl:UbcH7 complex, which is in fact an inactive pair. b) Addition of 0.5 molar eq. of E4BU^R1143A to ¹⁵N-UbcH5c-O~¹⁵N-Ub (orange) results in CSPs plotted in the histogram. 0.5 molar eq. titration points of E4BU^WT into free ¹⁵N-UbcH5c/Ub (cyan) and ¹⁵N-UbcH5c-O~¹⁵N-Ub (black) are shown for reference. c) E4BU⁺²⁰ auto-ubiquitination assays show that the E4B^R1143A mutant is deficient in Ub transfer activity. Gel slices taken from figure S4B.

Figure 5. Allosteric activation by RING/U-Box E3s involves three discrete structural requirements

Formation of an active E3:E2~Ub complex depends on three structural requirements: 1) A consensus E3:E2 interaction at E2 Helix 1, Loop 4, and Loop 7 (reported by CSPs highlighted in dark blue), 2) Formation of an intermolecular E3:E2 hydrogen bond that causes secondary effects permeating through the E2 3₁₀ helix (reported by CSPs highlighted in cyan), and 3) Overpopulation of closed E2~Ub conformations that result in additional changes surrounding the active site (reported by CSPs highlighted in red). Each requirement can be targeted with specific point mutations. Class “a” mutations such as UbcH5c Loop 7 A96D disrupt formation of the consensus E3:E2 interaction. The essential intermolecular hydrogen bond can be disrupted via a class “b” mutation such as E4B R1143A. Finally, formation of closed E2~Ub conformations can be blocked with class “c” mutations such as UbcH5c D87E, UbcH5c L104Q, or Ub I44A.