Capturing a substrate in an activated RING E3/E2-SUMO complex

Abstract

Post-translational protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as small ubiquitin-like modifier (SUMO) regulates processes including protein homeostasis, the DNA damage response, and the cell cycle. Proliferating cell nuclear antigen (PCNA) is modified by Ub or poly-Ub at lysine (Lys)164 after DNA damage to recruit repair factors. Yeast PCNA is modified by SUMO on Lys164 and Lys127 during S-phase to recruit the anti-recombinogenic helicase Srs2. Lys164 modification requires specialized E2/E3 enzyme pairs for SUMO or Ub conjugation. For SUMO, Lys164 modification is strictly dependent on the E3 ligase Siz1, suggesting the E3 alters E2 specificity to promote Lys164 modification. The structural basis for substrate interactions in activated E3/E2–Ub/Ubl complexes remains unclear. Here we report an engineered E2 protein and cross-linking strategies that trap an E3/E2–Ubl/substrate complex for structure determination, illustrating how an E3 can bypass E2 specificity to force-feed a substrate lysine into the E2 active site.

Extended Data Figure 1. E2_Ubc9-SUMO Thioester Mimic and Cross-Linking to Substrate PCNA for Reconstitution With E3_Siz1

a, SDS-PAGE analysis of in vitro E2_Ubc9^A129K or E2_Ubc9^C93K charging with SUMO in the presence and absence of E3_Siz1^(167–465) at pH (7.5) (left) and purification of the E2_Ubc9^C93K-SUMO (middle) and E2_Ubc9^A129K-SUMO (right) thioester mimetics. b, Plots of rates for in vitro SUMO modification of PCNA in assays utilizing various concentrations of purified E2_Ubc9-SUMO^D68R-Alexa488 labeled thioester, 1 nM E3_Siz1^(167–465) and 32 µM PCNA with 0, 2, 5 or 20 µM of the E2_Ubc9^C93K-SUMO or E2_Ubc9^A129K-SUMO thioester mimic (left) with exemplary non-reducing SDS-PAGE for the 0.5 µM E2_Ubc9-SUMO^D68R-Alexa488 reactions (right). The calculated K_m and K_i from these fits are shown in Extended Data Tables 1a and 2 and the quantified data show mean ± s.d. (n=3 technical replicates). c, SDS-PAGE analysis (left) of numbered 0.5 ml fractions from Superose6 analytical gel-filtration analysis (right) of complex reconstitution between E2_Ubc9-SUMO-BMOE-PCNA and E3_Siz1^(167–465) (green) or the E3_Siz1^(167–465)-SUMO fusion (blue). Elution profiles for E2_Ubc9-SUMO-BMOE-PCNA (purple) and E3_Siz1^(167–465) (red) alone are shown. d, Plot of the normalized change in polarization observed upon addition of serially diluted E2_Ubc9 with Alexa488 labeled SUMO or SUMO^D68R. Data were fit to single-site binding model accounting for receptor depletion. Data show mean ± s.d. (n=3 technical replicates). For gel source data, see Supplementary Figure 1.

Extended Data Figure 2. Comparing Strategies for Crosslinking the E2-SUMO Thioester Mimic and Substrate PCNA

a, Chemical structures of the proposed tetrahedral intermediate formed during PCNA Lys164 attack of E2_Ubc9-SUMO thioester (left), a BMOE cross-link (middle) or an EDT cross-link (right) between E2_Ubc9-SUMO C93 and PCNA K164C. Indicated distances were estimated with ChemDraw15 (PerkinElmer). b, Control non-reducing SDS-PAGE panel for Fig. 1a showing mock treated PCNA K127R/K164C (DMSO instead of EDT in DMSO) is unable to accept transthioesterification of SUMO at position 164. c, SDS-PAGE analysis of the 5 ml fractions from the final preparative Superdex200 gel-filtration purification of the E2_Ubc9-SUMO-EDT-PCNA/ E3_Siz1^(167–449)-SUMO complex. For gel source data, see Supplementary Figure 1.

Extended Data Figure 3. E2_Ubc9 Active Site, Conformation of SUMO^D and Comparison to Relevant Structures

a, Stereo image of simulated annealing electron density map showing the EDT linkage and the SUMO Gly98 linkage to E2_Ubc9 A129K. The 2F_o-Fc electron density map is contoured at 0.8σ (grey mesh). b, Alignment of the E2s from the current structure, SUMO modified RanGAP1 bound to E2_Ubc9^K14R and E3_Znf451 (5D2M), E2_Ubc5B^{S22R/N77A/C85S}-Ub bound to the RING dimer from E3_BIRC7 (4AUQ) and E2 E2_Ubc5A^S22R/C85K-Ub bound to the RING dimer from E3_RNF4 (4AP4) showing two orientations of the E2 active site. c, Model of tetrahedral intermediate generated by comparing our structure to other structures of E2-Ubl/E3 complexes, particularly Protein Data Bank (PDB) 5DM2 and 4P5O. d, Alignment of the current structure and three E2/RING (1UR6, 3EB6, and 3FN1) complexes and one E2/UBox (2C2V) complex (aligned by the E2). e, Alignments of four E2-Ubls/E3 complexes (aligned by the E2) in the closed activated confirmation for the current structure, E2_Ubc9 ^K14R -SUMO (5D2M), E2_Ubc5A^S22R/C85K-Ub (4AP4) and E2_Ubc12^N103S/C111S-Nedd8 (4P5O). f, SDS-PAGE analysis of multiple turnover assays of SUMO modification of PCNA utilizing in vitro reactions with coupled E1 (200 nM), E2 (100 nM), and E3 (50 nM) activities with 4 µM PCNA for the quantified data shown in Fig. 2c. g, Alignments of the E2 from relevant structures with lysine or arginine residues within or projecting toward the E2 active sites compared to the current structure. Lysine 63 from acceptor ubiquitin projecting toward the active site of the E2_Ubc13^C87S-Ub is shown in green (2GMI). Lysine 524 from SUMO modified RanGAP1 laying across the active site of E2_Ubc9^K14R is shown in magenta. The Lys720Arg from Cullin-1 projecting into the active site of E2_Ubc12-Nedd8 is shown in grey (4P5O). For the current structure, EDT was removed from the model, Cys164 was mutated back to lysine and the side chain was fit to the electron density and is shown in pink in reference to the current E2 (blue) and donor SUMO (orange). For gel source data, see Supplementary Figure 1.

Extended Data Figure 4. SUMO^B Bound to the E2 Backside enhances E2_Ubc9-SUMO Recruitment

a, Alignment of the current E2_Ubc9/backside SUMO^B (left) to previously observed E2_Ubc9/backside SUMO complexes (right). The position of the D68R mutation is shown in red sticks (left). b, Primary E3_Siz1 structure (top). Cartoons indicating the E3_Siz1 or E3_Siz1-SUMO fusion constructs utilized in the multiple turnover in vitro assays (middle) shown in Fig. 3 utilizing a titration of the purified E2_Ubc9-SUMO^D68R-Alexa488 thioester with or without 1.5-fold excess of the indicated additional molecule of non-conjugatable SUMO, 1 nM of the indicated E3 construct and 32 µM PCNA. Representative non-reducing SDS-PAGE showing the 0.5 µM E2_Ubc9-SUMO^D68R-Alexa488 thioester reactions below the plots of the rates of reaction for each E2_Ubc9-SUMO^D68R concentration (middle). The kinetics of SUMO modification of PCNA were calculated and the K_m and k_cat were determined (bottom) and are also shown in Extended Data Table 2. The quantified rate data show mean ± s.d. (n=3 technical replicates). For gel source data, see Supplementary Figure 1.

Extended Data Figure 5. E2_Ubc9 and E3_Siz1 Determinants of Lysine Specificity

a. Plots of the rates observed at different pH values for multiple turnover in vitro assays of SUMO modification of PCNA utilizing 0.1 µM purified E2_Ubc9-SUMO^D68R-Alexa488 thioester (or E2_Ubc9 mutant thioesters) with 5 nM E3_Siz1 and 4 µM PCNA at 4°C. b, SDS-PAGE analysis of multiple turnover assays of SUMO modification of PCNA utilizing in vitro reactions with coupled E1 (200 nM), E2 (100 nM), and E3 (50 nM) activities with 4 µM PCNA for the quantified data shown in Fig. 4d. c, SDS-PAGE analysis of multiple turnover assays of SUMO modification of PCNA utilizing in vitro reactions with coupled E1 (200 nM), E2 (100 nM), and E3 (50 nM) activities with 4 µM PCNA and quantified. d, Representative non-reducing SDS-PAGE analysis of the single turnover in vitro assays of SUMO modification of PCNA shown in Fig. 4e. These assays utilize 5 nM of the E2_Ubc9-SUMO^D68R-Alexa488 thioester (or E2_Ubc9 mutant thioesters) in reactions with 50 nM of the indicated E3_Siz1 and a titration of PCNA. Shown are typical SDS-PAGE analyses from the 10 µM PCNA reactions. The data were used to extract the kinetic constants for the reactions shown as histograms and in Extended Data Table 4. For panels a,c and d the quantified rate data show mean ± s.d. (n=3 technical replicates). For gel source data, see Supplementary Figure 1.

Extended Data Figure 6. Shape Complementarity between the E2_Ubc9-SUMO/E3 Complex and PCNA

a, The current structure (color) with the crystallographic packing of a lattice mate PCNA molecule (black). b, Non-reducing SDS-PAGE analysis of 2 minute endpoint in vitro E2_Ubc9-SUMO thioester formation reactions with 0.05 µM E1, 0.4 µM of the indicated E2_Ubc9 and 22 µM SUMO (left) and the quantitated E2-SUMO band (right). The quantified band intensity shows mean ± s.d. (n=3 technical replicates). c, SDS-PAGE analysis of multiple turnover assays of SUMO modification of PCNA utilizing in vitro reactions with coupled E1 (200 nM), E2 (100 nM), and E3 (50 nM) activities with 4 µM PCNA or without PCNA (diSUMO formation) shown quantified in Fig. 5c. d, Location of E2_Ubc9 and PCNA mutations that had no effect (red sticks) on activities observed for in vitro assays similar to those performed in Fig. 5c in relation to residues that did show effects (green sticks). e, The E2_Ubc13^C87S-Ub was aligned to E2_Ubc9 in the current structure and subsequently the Lys164/Glu165 loop from trimeric PCNA (pink) was aligned onto the Lys63/Glu64 loop from acceptor ubiquitin (2GMI, green). Within this conformation the E3_Siz1 PINIT domain (cyan) clashes with another protomer of the PCNA trimer (grey). For gel source data, see Supplementary Figure 1.

Figure 1. Reconstituting E2_Ubc9-SUMO^D/E3_Siz1-SUMO^B/PCNA

a, Schematic for nucleophilic attack of the E2-SUMO thioester by lysine or EDT modified cysteine with in vitro SUMO modification of PCNA. b, Schematic of the tetrahedral intermediate during transthioesterification and EDT cross-linking of E2_Ubc9^A129K-SUMO and PCNA. c, Structure of the complex. d, Cartoon model of the complex. For gel source data, see Supplementary Figure 1.

Figure 2. E3 Activation of E2_Ubc9-SUMO^D

a, E2/SP-RING interactions (left) and the structure with PINIT removed (right). b, SP-CTD/SUMO^D interactions (left) and overview (right). c, Quantification of multiple turnover assays of SUMO modification of PCNA with coupled E1, E2, and E3 activities. Quantified rate data show mean ± s.d. (n=3 technical replicates).

Figure 3. SUMO^B Aids in E2_Ubc9-SUMO^D Recruitment

Specific activities for E3 and E3-SUMO fusion construct-catalyzed multiple turnover reactions with E2-SUMO^D68R thioester titrations with and without 1.5-fold excess SUMO. Quantified rate data show mean ± s.d. (n=3 technical replicates).

Figure 4. E3/PCNA interactions and Lysine Specificity

a, E3_Siz1 PINIT/PCNA interactions. b, E2 active site interactions with the FKS loop from the E3_Siz1 PINIT domain (EDT in green). c, Comparison of the E2_Ubc9 active sites with PCNA or RanGAP1. d, Quantification of multiple turnover assays for SUMO modification of PCNA with coupled E1, E2 and E3 activities. e, Kinetics of single turnover assays with E2_Ubc9-SUMO^D68R thioester, E3 and PCNA. For panels d and e, quantified data show mean ± s.d. (n=3 technical replicates).

Figure 5. Surface Complementarity between E2_Ubc9-SUMO^D/E3 and PCNA

a, Trimeric PCNA (black ribbon and grey cartoon) modeled onto the current structure (color). b, Location of E2 and PCNA residues with detrimental effects on activity. c, Multiple turnover assays of SUMO modification of PCNA with coupled E1, E2, and E3 activities. Quantified data show mean ± s.d. (n=3 technical replicates). d, Model of E2-SUMO/E3/Trimeric PCNA indicating the site for polymerase opposite the presumed position of the N-terminal E3_Siz1 SAP domain.