Abstract
Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.
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References
Pickart, C.M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).
Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).
Chen, Z. & Pickart, C.M. A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin. J. Biol. Chem. 265, 21835–21842 (1990).
Brzovic, P.S. & Klevit, R.E. Ubiquitin transfer from the E2 perspective: why is UbcH5c so promiscuous? Cell Cycle 5, 2867–2873 (2006).
Nuber, U., Schwarz, S., Kaiser, P., Schneider, R. & Scheffner, M. Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2–F1) and characterization of their interaction E6-AP and RSP5. J. Biol. Chem. 271, 2795–2800 (1996).
Machida, Y.J. et al. UBE2T is the E2 in the Fanconi anemia pathway and undergoes negative autoregulation. Mol. Cell 23, 589–596 (2006).
McDowell, G.S., Kucerova, R. & Philpott, A. Non-canonical ubiquitylation of the proneural protein Ngn2 occurs in both Xenopus embryos and mammalian cells. Biochem. Biophys. Res. Commun. 400, 655–660 (2010).
Vosper, J.M. et al. Ubiquitylation on canonical and non-canonical sties targets the transcription factor neurogenin for ubiquitin-mediate proteolysis. J. Biol. Chem. 284, 15458–15468 (2009).
Scaglione, K.M. et al. The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N terminus of substrates. J. Biol. Chem. 288, 18784–18788 (2013).
Tatham, M.H., Plechanovová, A., Jaffray, E.G., Salmen, H. & Hay, R.T. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini. Biochem. J. 453, 137–145 (2013).
Wu, W. et al. BRCA1 ubiquitinates RPB8 in response to DNA damage. Cancer Res. 67, 951–958 (2007).
Christensen, D.E., Brzovic, P.S. & Klevit, R.E. E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nat. Struct. Mol. Biol. 14, 941–948 (2007).
Guzzo, C.M. et al. RNF4-dependent hybrid SUMO-ubiquitin chains are signals for RAP80 and thereby mediate the recruitment of BRCA1 to site of DNA damage. Sci. Signal. 5, ra88 (2012).
Wenzel, D.M., Lissounov, A., Brzovic, P.S. & Klevit, R.E. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105–108 (2011).
Zhang, Y. et al. UBE2W interacts with FANCL and regulates the monoubiquitination of Fanconi anemia protein FANCD2. Mol. Cells 31, 113–122 (2011).
Alpi, A.F., Pace, P.E., Babu, M.M. & Patel, K.J. Mechanistic insight into site-restricted monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI. Mol. Cell 32, 767–777 (2008).
Wand, A.J., Urbauer, J.L., McEvoy, R.P. & Beiber, R.J. Internal dynamics of human ubiquitin revealed by 13C-relaxation studies of randomly fractionally labeled protein. Biochemistry 35, 6116–6125 (1996).
Berman, H.M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).
Kelley, L.A. & Sternberg, M.J.E. Protein structure prediction on the web: a case study using the Phyre server. Nat. Protoc. 4, 363–371 (2009).
Jones, D.T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202 (1999).
Plechanovová, A., Jaffray, E.G., Tatham, M.H., Naismith, J.H. & Hay, R.T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012).
Grimsley, G.R., Scholtz, J.M. & Pace, C.N. A summary of the measured pK values of the ionizable groups in folded proteins. Protein Sci. 18, 247–251 (2009).
Sheng, Y. et al. A human ubiquitin conjugating enzyme (E2)-HECT E3 ligase structure-function screen. Mol. Cell. Proteomics 11, 329–341 (2012).
Vittal, V., Wenzel, D.M., Brzovic, P.S. & Klevit, R.E. Biochemical and structural characterization of the ubiquitin-conjugating enzyme UBE2W reveals the formation of a noncovalent homodimer. Cell Biochem. Biophys. 67, 103–110 (2013).
Laskowski, R.A. PDBsum new things. Nucleic Acids Res. 37, D355–D359 (2009).
Shen, Y. et al. Consistent blind protein structure generation from NMR chemical shit data. Proc. Natl. Acad. Sci. USA 105, 4685–4690 (2008).
Shen, Y., Vernon, R., Baker, D. & Bax, A. De novo protein structure generation from incomplete chemical shift assignments. J. Biol. NMR 43, 63–78 (2009).
Pruneda, J.N. et al. Structure of an E3:E2∼Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol. Cell 47, 933–942 (2012).
Dou, H., Buetow, L., Sibbet, G.J., Cameron, K. & Huang, D.T. BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nat. Struct. Mol. Biol. 19, 876–883 (2012).
Wu, P.Y. et al. A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J. 22, 5241–5250 (2003); erratum 23, 4876 (2004); erratum 26, 4051 (2007).
Berndsen, C.E., Wiener, R., Yu, I.W., Ringel, A.E. & Wolberger, C. A conserved asparagine has a structural role in ubiquitin-conjugating enzyme. Nat. Chem. Biol. 9, 154–156 (2013).
Hershko, A., Heller, H., Eytan, E., Kaklij, G. & Rose, I.A. Role of the α-amino group of protein in ubiquitin-mediated protein breakdown. Proc. Natl. Acad. Sci. USA 81, 7021–7025 (1984).
Breitschopf, K., Bengal, E., Ziz, T., Admon, A. & Ciechanover, A. A novel site of ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. EMBO J. 17, 5964–5973 (1998).
Ciechanover, A. & Ben-Saadon, R. N-terminal ubiquitination: more protein substrates join in. Trends Cell Biol. 14, 103–106 (2004).
Coulombe, P., Rodier, G., Bonneil, E., Thibault, P. & Meloche, S. N-terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome. Mol. Cell. Biol. 24, 6140–6150 (2004).
Dormeyer, W., Mohammed, S., Breukelen, B., Krijgsveld, J. & Heck, A.J. Targeted analysis of protein termini. J. Proteome Res. 6, 4634–4645 (2007).
Yin, G. et al. Cloning, characterization and subcellular localization of a gene encoding a human ubiquitin-conjugating enzyme (E2) homologous to the Arabidopsis thaliana UBC-16 gene product. Front. Biosci. 11, 1500–1507 (2006).
Pickart, C.M. & Raasi, S. Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005).
Brzovic, P.S. et al. Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex. Proc. Natl. Acad. Sci. USA 100, 5646–5651 (2003).
Todi, S.V. et al. Cellular turnover of the polyglutamine disease protein ataxin-3 is regulated by its catalytic activity. J. Biol. Chem. 282, 29348–29358 (2007).
Barghorn, S., Biernat, J. & Mandelkow, E. Purification of recombinant tau protein and preparation of Alzheimer-paired helical filaments in vitro. Methods Mol. Biol. 299, 35–51 (2005).
Winborn, B.J. et al. The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains. J. Biol. Chem. 283, 26436–26443 (2008).
Xu, Z. et al. Structure and interactions of the helical and U-box domains of CHIP, the C terminus of HSP70 interacting protein. Biochemistry 45, 4749–4759 (2006).
Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158 (1999).
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
Johnson, B.A. & Blevins, R.A. NMR View: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).
Svergun, D., Barberato, C. & Koch, M.H.J. CRYSOL: A program to evaluate X-ray solution scatter of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995).
Raman, S. et al. NMR structure determination for larger proteins using backbone-only data. Science 327, 1014–1018 (2010).
Thompson, J.M. et al. Accurate protein structure modeling using sparse NMR data and homologous structure information. Proc. Natl. Acad. Sci. USA 109, 9875–9880 (2012).
Bhattacharya, A., Tejero, R. & Gaetano, M.T. Evaluating protein structures determined by structural genomics consortia. Proteins 66, 778–795 (2007).
Ulmer, T.S., Ramirez, B.E., Delaglio, F. & Bax, A. Evaluation of backbone proton positions and dynamics in a small protein by liquid crystal NMR spectroscopy. J. Am. Chem. Soc. 125, 9179–9191 (2003).
Acknowledgements
We acknowledge D. Christensen and C. Eakin for their initial observations on Ube2w and J.N. Pruneda for collecting SAXS data on Ube2w. This work was supported by National Institute of General Medical Sciences grants R01 GM088055 (REK), R01 GM098503 (P.S.B.), K99 NS073936 (K.M.S.) and R01 AG034228 (H.L.P.) and the University of Washington Hurd Fellowship Fund and Public Health Service National Research Service Award 2T32 GM007270 (to V.V.).
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V.V., P.S.B. and R.E.K. conceived the experiments and wrote the manuscript. V.V. performed the biochemical and structural experiments with help from K.M.S. and E.D.D. D.M.W. performed the initial characterization of Ube2w. V.B. and K.S.J.E.-J. performed MS. L.S. and D.B. performed the structure calculations. H.L.P. provided guidance. R.E.K. supervised the project.
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Supplementary Results, Supplementary Figures 1–16 and Supplementary Tables 1–3. (PDF 21157 kb)
Supplementary Data Set 1
Raw experimental RDC values for anisotropic and isotropic N-H spectra collected with and without Pf1 phage are provided. (XLSX 53 kb)
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Vittal, V., Shi, L., Wenzel, D. et al. Intrinsic disorder drives N-terminal ubiquitination by Ube2w. Nat Chem Biol 11, 83–89 (2015). https://doi.org/10.1038/nchembio.1700
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DOI: https://doi.org/10.1038/nchembio.1700
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