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

Nature volume 536, pages 304–308 (2016)Cite this article

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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.

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Figure 1: Reconstituting E2Ubc9–SUMOD/E3Siz1–SUMOB/PCNA.
Figure 2: E3 Activation of E2Ubc9–SUMOD.
Figure 3: SUMOB Aids in E2Ubc9–SUMOD Recruitment.
Figure 4: E3/PCNA interactions and lysine specificity.
Figure 5: Surface complementarity between E2Ubc9–SUMOD/E3 and PCNA.

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Protein Data Bank

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Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession number 5JNE.

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Acknowledgements

Research at NE-CAT beamlines was funded by P41 GM103403 (National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)), S10 RR029205 (NIH-ORIP (Office of Research Infrastructure Programs) High-End Shared Instrument grant) at the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory (DE-AC02-06CH11357). Research was supported in part by GM065872 and GM118080 (NIH/NIGMS, C.D.L.) and P30CA008748 (NIH/ National Cancer Institute). The content is the authors’ responsibility and does not represent the official views of the NIH. C.D.L. is a Howard Hughes Medical Institute Investigator.

Author information

Authors and Affiliations

  1. Structural Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, 10065, New York, USA

    Frederick C. Streich Jr & Christopher D. Lima

  2. Howard Hughes Medical Institute, 1275 York Avenue, New York, 10065, New York, USA

    Christopher D. Lima

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  1. Frederick C. Streich Jr
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F.C.S. and C.D.L. executed experiments, data analysis, and manuscript preparation.

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Correspondence to Christopher D. Lima.

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Reviewer Information Nature thanks B. Eichman, R. Hay and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 E2Ubc9–SUMO thioester mimic and cross-linking to substrate PCNA for reconstitution with E3Siz1.

a, SDS–PAGE analysis of in vitro or charging with SUMO in the presence and absence of at pH (7.5) (left) and purification of the –SUMO (middle) and –SUMO (right) thioester mimetics. b, Rates for in vitro SUMO modification of PCNA in assays using various concentrations of purified E2Ubc9–SUMOD68R–Alexa488-labelled thioester, 1 nM , and 32 μM PCNA with 0, 2, 5, or 20 μM of the –SUMO or –SUMO thioester mimic (left) with exemplary non-reducing SDS–PAGE for the 0.5 μM E2Ubc9–SUMOD68R–Alexa488 reactions (right). The calculated Km and Ki from these fits are shown in Extended Data Tables 1 and 3 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 E2Ubc9–SUMO–BMOE–PCNA and (green) or the –SUMO fusion (blue). Elution profiles for E2Ubc9–SUMO–BMOE–PCNA (purple) and (red) alone are shown. d, The normalized change in polarization observed upon addition of serially diluted E2Ubc9 with Alexa488 labelled SUMO or SUMOD68R. Data were fitted to a single-site binding model accounting for receptor depletion. Data show mean ± s.d. (n = 3 technical replicates). For gel source data, see Supplementary Fig. 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 E2Ubc9–SUMO thioester (left), a BMOE cross-link (middle) or an EDT cross-link (right) between E2Ubc9–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 E2Ubc9–SUMO–EDT–PCNA/–SUMO complex. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 E2Ubc9 active site, conformation of SUMOD, and comparison with relevant structures.

a, Stereo image of simulated annealing electron density map showing the EDT linkage and the SUMO Gly98 linkage to E2Ubc9 A129K. The 2Fo − Fc electron density map is contoured at 0.8σ (grey mesh). b, Alignment of the E2 enzymes from the current structure, SUMO-modified RanGAP1 bound to and E3Znf451 (5D2M), -Ub bound to the RING dimer from E3BIRC7 (4AUQ), and E2 -Ub bound to the RING dimer from E3RNF4 (4AP4) showing two orientations of the E2 active site. c, Model of tetrahedral intermediate generated by comparing our structure with other structures of E2–Ubl/E3 complexes, particularly Protein Data Bank (PDB) accession numbers 5DM2 and 4P5O. d, Alignment of the current structure and three E2/RING (PDB 1UR6, 3EB6, and 3FN1) complexes and one E2/UBox (PDB 2C2V) complex (aligned by the E2). e, Alignments of four E2–Ubl/E3 complexes (aligned by the E2) in the closed activated confirmation for the current structure, –SUMO (PDB 5D2M), -Ub (PDB 4AP4), and -Nedd8 (PDB 4P5O). f, SDS–PAGE analysis of multiple turnover assays of SUMO modification of PCNA using 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 E2 from relevant structures with lysine or arginine residues within or projecting towards the E2 active sites compared with the current structure. Lysine 63 from acceptor ubiquitin projecting towards the active site of the –Ub is shown in green (PDB 2GMI). Lysine 524 from SUMO-modified RanGAP1 laying across the active site of is shown in magenta. The Lys720Arg from Cullin-1 projecting into the active site of E2Ubc12–Nedd8 is shown in grey (PDB 4P5O). For the current structure, EDT was removed from the model, Cys164 was mutated back to lysine, and the side chain was fitted 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 Fig. 1.

Extended Data Figure 4 SUMOB bound to the E2 backside enhances E2Ubc9–SUMO recruitment.

a, Alignment of the current E2Ubc9/backside SUMOB (left) to previously observed E2Ubc9/backside SUMO complexes (right). The position of the D68R mutation is shown in red sticks (left). b, Primary E3Siz1 structure (top). Cartoons indicating the E3Siz1 or E3Siz1–SUMO fusion constructs used in the multiple turnover in vitro assays (middle) shown in Fig. 3 using a titration of the purified E2Ubc9–SUMOD68R–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 E2Ubc9–SUMOD68R–Alexa488 thioester reactions below the plots of the rates of reaction for each E2Ubc9–SUMOD68R concentration (middle). The kinetics of SUMO modification of PCNA were calculated and Km and kcat determined (bottom); these are shown in Extended Data Table 3. The quantified rate data show mean ± s.d. (n = 3 technical replicates). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 5 E2Ubc9 and E3Siz1 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 using 0.1 μM purified E2Ubc9–SUMOD68R–Alexa488 thioester (or E2Ubc9 mutant thioesters) with 5 nM E3Siz1 and 4 μM PCNA at 4 °C. b, SDS–PAGE analysis of multiple turnover assays of SUMO modification of PCNA using 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 using 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 utilized 5 nM of the E2Ubc9–SUMOD68R–Alexa488 thioester (or E2Ubc9 mutant thioesters) in reactions with 50 nM of the indicated E3Siz1 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 5. For a, c, and d the quantified rate data show mean ± s.d. (n = 3 technical replicates). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 6 Shape complementarity between the E2Ubc9–SUMO/E3 complex and PCNA.

a, The current structure (colour) with the crystallographic packing of a lattice mate PCNA molecule (black). b, Non-reducing SDS–PAGE analysis of 2 min endpoint in vitro E2Ubc9–SUMO thioester formation reactions with 0.05 μM E1, 0.4 μM of the indicated E2Ubc9, and 22 μM SUMO (left) and the quantified 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 using 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 E2Ubc9 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 –Ub was aligned to E2Ubc9 in the current structure and subsequently the Lys164/Glu165 loop from trimeric PCNA (pink) was aligned onto the Lys63/Glu64 loop from acceptor ubiquitin (PDB 2GMI, green). Within this conformation the E3Siz1 PINIT domain (cyan) clashes with another protomer of the PCNA trimer (grey). For gel source data, see Supplementary Fig. 1.

Extended Data Table 1 Summary of kinetic constants or inhibition constants for E2Ubc9–SUMO thioester and thioester mimetic association to E3Siz1
Full size table
Extended Data Table 2 Summary of binding curve fits of fluorescent polarization data for Ubc9 binding Alexa488-labelled SUMO or SUMOD68R
Full size table
Extended Data Table 3 Summary of kinetic constants for multiple turnover experiments with purified E2Ubc9–SUMOD68R thioester
Full size table
Extended Data Table 4 Data collection and refinement statistics
Full size table
Extended Data Table 5 Summary of kinetic constants for single turnover experiments with purified E2Ubc9–SUMOD68R thioester
Full size table

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Streich Jr, F., Lima, C. Capturing a substrate in an activated RING E3/E2–SUMO complex. Nature 536, 304–308 (2016). https://doi.org/10.1038/nature19071

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