Abstract
E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. Here, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. The ZNF451 catalytic module contains tandem SUMO-interaction motifs (SIMs) bridged by a Pro-Leu-Arg-Pro (PLRP) motif. The first SIM and PLRP motif engage thioester-charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the back side of E2. We show that ZNF451 is SUMO2 specific and that SUMO modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase.
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Acknowledgements
We would like to thank A.P.'s laboratory for providing the pLou3-ZNF4512–56 and pLou3-ZNF4512–247 plasmids, and A.P.'s and C.D.L.'s labs for discussion. Data for this study were measured at beamline X29 of the National Synchrotron Light Source, whose financial support comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy (DOE), the National Center for Research Resources (P41RR012408) and the National Institute of General Medical Sciences (NIGMS; P41GM103473) of the US National Institutes of Health (NIH). This work is also based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the NIGMS from the NIH (P41 GM103403). The Pilatus 6M detector on the 24-ID-C beamline is funded by a NIH Office of Research Infrastructure Programs High End Instrumentation grant (S10 RR029205). This research used resources of the Advanced Photon Source, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Research reported in this publication was supported in part by the NIGMS of the NIH under award number GM065872 (C.D.L.) and the NIH–National Cancer Institute Cancer Center Support Grant P30 CA008748. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. A.P. is supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG-SPP1365 PI 917/2-1). L.C. is supported in part by a fellowship from the Fonds de Recherche du Québec–Santé. C.D.L. is supported as an Investigator of the Howard Hughes Medical Institute.
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A.P. initiated the project. L.C. and C.D.L. designed experiments that L.C. performed. L.C. and C.D.L. wrote the manuscript with input from A.P.
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Integrated supplementary information
Supplementary Figure 1 Noncovalent interaction between SUMO2 and UBC9
Fluorescence polarization assay performed using 50 nM Alexa488-labeled SUMO21–93 and serially diluted UBC9. Data is mean ± s.d. (n=3 technical replicates). Data was fitted to a single site binding model accounting for ligand depletion. The assay was performed in 20 mM HEPES pH 7.5, 50 mM NaCl and 0.1 % Tween-20.
Supplementary Figure 2 Electron density maps and analysis of the protein-protein contacts in the asymmetric unit.
(a) Stereo view of the electron density map around the ZNF451 PLRP motif. Protein is in stick representation with red spheres representing water molecules. ZNF451 is colored in green, SUMO2D in yellow and SUMO2B in orange. The simulated annealing composite omit 2mFo – DFc map is contoured at 1.5 σ and shown as a grey mesh. (b) Surface representation of the proteins in the asymmetric unit of the ZNF451–SUMO2~RANGAP1–UBC9 complex. Selected interface areas are presented and highlighted using arrows and filled circles. (c) Table generated with PISA (Krissinel, E. & Henrick, K. J Mol Biol 372, 774-97, 2007) showing the list of interactions in the crystal lattice. ΔiG is the solvation free energy gain upon formation of the interface. NHB and NSB are the numbers of hydrogen bonds and salt-bridges across the interface. Only interfaces burying more than 100 Å2 of accessible surface area are listed.
Supplementary Figure 3 Structural comparison of the SUMO-SIM interactions and the PLRP motif.
SUMO–SIM interactions at the (a) SUMO2D–ZNF451, (b) SUMO2D–RANBP2 (pdb 3UIN; Gareau, J.R et al., J Biol Chem 287, 4740-51, 2012), or (c) SUMO2B–ZNF451 interfaces. The left panels highlight the SUMO2 residues that interact with the SIMs. SUMO2 is in surface representation. The SUMO2 residues interacting with the SIMs are presented in stick representation. Residues belonging to ZNF451 and RANBP2 are also in stick representation. The right panels highlight the hydrogen bond and salt bridges interactions involved in the SUMO–SIM interface. For simplicity, only the side-chains establishing hydrogen bonds or salt bridges interactions are presented. Grey dashes and bold numbers represent hydrogen bonds and their length. The view was rotated by 45° along the x axis as compared to the left panels. (d) Structural similarity between the PLRP motif of ZNF451 and the one present in other proteins. Structures containing a PLRP sequence were selected and the PLRP sequence of each protein was aligned to the one of ZNF451. Only structural alignment resulting in a rmsd < 1 Å are presented. Also, only one representative structure per protein is presented. The pdb accession numbers are provided for each of the structures.
Supplementary Figure 4 Effect of the ZNF4512–55-SUMO215–89 fusion on the conjugation rates of SUMO2 to the p53 tetramerization domain.
Single-turnover conjugation rates of SUMO2 to p53 tetramerization domain (residues 320-393) in presence of 100 nM fusion ZNF4512–55SUMO215–89 or ZNF4512–56. Left, plot showing the initial rate of SUMO conjugation versus p53 concentration. Data show mean ± s.d. (n=3 technical replicates). Data for ZNF4512–56 is the same as in Figure 1b. Right, representative SDS-PAGE for SUMO2 conjugation to p53 in presence of 100 nM fusion ZNF4512–55SUMO215–89. Assays were done at 0°C in 20 mM HEPES pH 7.5, 50 mM NaCl, 0.1 % Tween-20 and 5 mM EDTA using 28 ± 5 nM thioester charged UBC9~Alexa488-labeled SUMO2, 100 nM fusion ZNF4512–55SUMO215–89 and serially diluted p53 at the indicated concentration.
Supplementary Figure 5 Purification of SUMO215–93~Znf4512–110
12 % NuPAGE gel run with MES buffer and stained with Coommassie Brillant Blue showing the purification of SUMO215–93-Znf4512–110. Conjugation reaction was performed for 1h and passed on a HiPrep Desalting column equilibrated with 20 mM Tris pH 8, 50 mM NaCl and 1 mM 2-mercaptoethanol. The desalted reaction was then applied on a MonoQ column and eluted with a 50 to 1,000 mM NaCl gradient. Fractions 32 and 33 were pooled and used in lysine discharge assays.
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Supplementary Data Set 1
Uncropped gel and representative gels related to main figures (PDF 1941 kb)
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Cappadocia, L., Pichler, A. & Lima, C. Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase. Nat Struct Mol Biol 22, 968–975 (2015). https://doi.org/10.1038/nsmb.3116
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DOI: https://doi.org/10.1038/nsmb.3116
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