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Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer

Abstract

The ubiquitin–proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.

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Fig. 1: Stapled peptides modeled after Ubc15 helix 1 inhibit E1-mediated thioester transfer of ubiquitin to E2.
Fig. 2: Biophysical and biochemical properties of an optimized stapled peptide mimetic of E2h1 that binds and inhibits E1.
Fig. 3: Sequence determinants for SAH-UBE2A inhibition of E1.
Fig. 4: Influence of SAH-UBE2A on the conformational dynamics of E1.
Fig. 5: SAH-UBE2A is a pan-inhibitor of ubiquitin E1 thioester transfer that targets native E1 to achieve blockade of protein ubiquitination.

Data availability

All data generated or analyzed during this study are included in this manuscript and its supplementary information. HXMS data have been deposited to the PRIDE database with identifier PXD016014. Structures corresponding to PDB 6DC6, 5KNL, 4II2, 4II3 and 3CMM were used in this study. Source data are provided with this paper.

References

  1. Ciechanover, A., Elias, S., Heller, H. & Hershko, A. “Covalent affinity” purification of ubiquitin-activating enzyme. J. Biol. Chem. 257, 2537–2542 (1982).

    CAS  PubMed  Article  Google Scholar 

  2. Hershko, A., Heller, H., Elias, S. & Ciechanover, A. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206–8214 (1983).

    CAS  PubMed  Article  Google Scholar 

  3. Hershko, A., Leshinsky, E., Ganoth, D. & Heller, H. ATP-dependent degradation of ubiquitin-protein conjugates. Proc. Natl Acad. Sci. USA 81, 1619–1623 (1984).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. Hough, R., Pratt, G. & Rechsteiner, M. Purification of two high molecular weight proteases from rabbit reticulocyte lysate. J. Biol. Chem. 262, 8303–8313 (1987).

    CAS  PubMed  Article  Google Scholar 

  5. Popovic, D., Vucic, D. & Dikic, I. Ubiquitination in disease pathogenesis and treatment. Nat. Med. 20, 1242–1253 (2014).

    CAS  Article  PubMed  Google Scholar 

  6. Nalepa, G., Rolfe, M. & Harper, J. W. Drug discovery in the ubiquitin–proteasome system. Nat. Rev. Drug Discov. 5, 596–613 (2006).

    CAS  PubMed  Article  Google Scholar 

  7. Kane, R. C., Farrell, A. T., Sridhara, R. & Pazdur, R. United States Food and Drug Administration approval summary: bortezomib for the treatment of progressive multiple myeloma after one prior therapy. Clin. Cancer Res. 12, 2955–2960 (2006).

    CAS  PubMed  Article  Google Scholar 

  8. Kane, R. C. et al. Bortezomib for the treatment of mantle cell lymphoma. Clin. Cancer Res. 13, 5291–5294 (2007).

    CAS  PubMed  Article  Google Scholar 

  9. Bianchi, G. et al. The proteasome load versus capacity balance determines apoptotic sensitivity of multiple myeloma cells to proteasome inhibition. Blood 113, 3040–3049 (2009).

    CAS  PubMed  Article  Google Scholar 

  10. Harper, J. W. & Bennett, E. J. Proteome complexity and the forces that drive proteome imbalance. Nature 537, 328–338 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Ruggero, D. Translational control in cancer etiology. Cold Spring Harb. Perspect. Biol. 5, a012336 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  12. Obeng, E. A. et al. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood 107, 4907–4916 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Zhuang, J. et al. Ubiquitin-activating enzyme inhibition induces an unfolded protein response and overcomes drug resistance in myeloma. Blood 133, 1572–1584 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Hyer, M. L. et al. A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment. Nat. Med. 24, 186–193 (2018).

    CAS  PubMed  Article  Google Scholar 

  15. Huang, X. & Dixit, V. M. Drugging the undruggables: exploring the ubiquitin system for drug development. Cell Res. 26, 484–498 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Xu, G. W. et al. The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma. Blood 115, 2251–2259 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Jin, J., Li, X., Gygi, S. P. & Harper, J. W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature 447, 1135–1138 (2007).

    CAS  PubMed  Article  Google Scholar 

  18. Gavin, J. M. et al. Mechanistic studies on activation of ubiquitin and di-ubiquitin-like protein, FAT10, by ubiquitin-like modifier activating enzyme 6, Uba6. J. Biol. Chem. 287, 15512–15522 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Zhao, B. et al. Inhibiting the protein ubiquitination cascade by ubiquitin-mimicking short peptides. Org. Lett. 14, 5760–5763 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Barghout, S. H. et al. Preclinical evaluation of the selective small-molecule UBA1 inhibitor, TAK-243, in acute myeloid leukemia. Leukemia 33, 37–51 (2018).

    PubMed  Article  CAS  Google Scholar 

  21. MLN7243 Study No. C33001 Clinical Study Report (Takeda Pharmaceuticals International, 2017).

  22. Krishnamurty, R. & Maly, D. J. Biochemical mechanisms of resistance to small-molecule protein kinase inhibitors. ACS Chem. Biol. 5, 121–138 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Misra, M. et al. Dissecting the specificity of adenosyl sulfamate inhibitors targeting the ubiquitin-activating enzyme. Structure 25, 1120–1129 (2017).

    CAS  PubMed  Article  Google Scholar 

  24. Walensky, L. D. & Bird, G. H. Hydrocarbon-stapled peptides: principles, practice, and progress. J. Med. Chem. 57, 6275–6288 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Meric-Bernstam, F. et al. Phase I trial of a novel stapled peptide ALRN-6924 disrupting MDMX- and MDM2-mediated inhibition of WT p53 in patients with solid tumors and lymphomas. J. Clin. Oncol. 35, 2505 (2017).

    Article  Google Scholar 

  26. Lv, Z. et al. S. pombe Uba1-Ubc15 structure reveals a novel regulatory mechanism of ubiquitin E2 activity. Mol. Cell 65, 699–714 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Williams, K. M. et al. Structural insights into E1 recognition and the ubiquitin-conjugating activity of the E2 enzyme Cdc34. Nat. Commun. 10, 3296 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  28. Olsen, S. K. & Lima, C. D. Structure of a ubiquitin E1-E2 complex: insights to E1-E2 thioester transfer. Mol. Cell 49, 884–896 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Haas, A. L., Bright, P. M. & Jackson, V. E. Functional diversity among putative E2 isozymes in the mechanism of ubiquitin-histone ligation. J. Biol. Chem. 263, 13268–13275 (1988).

    CAS  PubMed  Article  Google Scholar 

  30. Tokgoz, Z. et al. E1-E2 interactions in ubiquitin and Nedd8 ligation pathways. J. Biol. Chem. 287, 311–321 (2012).

    PubMed  Article  CAS  Google Scholar 

  31. Lv, Z., Williams, K. M., Yuan, L., Atkison, J. H. & Olsen, S. K. Crystal structure of a human ubiquitin E1-ubiquitin complex reveals conserved functional elements essential for activity. J. Biol. Chem. 293, 18337–18352 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Aldrich, C. et al. The ecstasy and agony of assay interference compounds. ACS Cent. Sci. 3, 143–147 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Escudero, S. et al. Dynamic regulation of long-chain fatty acid oxidation by a noncanonical interaction between the MCL-1 BH3 helix and VLCAD. Mol. Cell 69, 729–743 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Lehman, T. C., Hale, D. E., Bhala, A. & Thorpe, C. An acyl-coenzyme A dehydrogenase assay utilizing the ferricenium ion. Anal. Biochem. 186, 280–284 (1990).

    CAS  PubMed  Article  Google Scholar 

  35. Engen, J. R. Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS. Anal. Chem. 81, 7870–7875 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Siepmann, T. J., Bohnsack, R. N., Tokgöz, Z., Baboshina, O. V. & Haas, A. L. Protein interactions within the N-end rule ubiquitin ligation pathway. J. Biol. Chem. 278, 9448–9457 (2003).

    CAS  PubMed  Article  Google Scholar 

  37. Lv, Z. et al. Domain alternation and active site remodeling are conserved structural features of ubiquitin E1. J. Biol. Chem. 292, 12089–12099 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Hann, Z. S. et al. Structural basis for adenylation and thioester bond formation in the ubiquitin E1. Proc. Natl Acad. Sci. USA 116, 15475–15484 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. Morgan, C. R. & Engen, J. R. Investigating solution-phase protein structure and dynamics by hydrogen exchange mass spectrometry. Curr. Protoc. Protein Sci. 58, 17.6.1–17.6.17 (2009).

    Google Scholar 

  40. Marcsisin, S. R. & Engen, J. R. Hydrogen exchange mass spectrometry: what is it and what can it tell us? Anal. Bioanal. Chem. 397, 967–972 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Lee, I. & Schindelin, H. Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell 134, 268–278 (2008).

    CAS  PubMed  Article  Google Scholar 

  42. Schäfer, A., Kuhn, M. & Schindelin, H. Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules. Acta Crystallogr. D Biol. Crystallogr. 70, 1311–1320 (2014).

    PubMed  Article  CAS  Google Scholar 

  43. Ding, Q. et al. Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in clinical development. J. Med. Chem. 56, 5979–5983 (2013).

    CAS  PubMed  Article  Google Scholar 

  44. Winter, G. E. et al. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Adams, J. Proteasome inhibition: a novel approach to cancer therapy. Trends Mol. Med. 8, S49–S54 (2002).

    CAS  PubMed  Article  Google Scholar 

  46. Lv, Z. et al. Molecular mechanism of a covalent allosteric inhibitor of SUMO E1 activating enzyme. Nat. Commun. 9, 5145 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. Bernal, F. et al. A stapled p53 helix overcomes HDMX-mediated suppression of p53. Cancer Cell 18, 411–422 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Cohen, N. A. et al. A competitive stapled peptide screen identifies a selective small molecule that overcomes MCL-1-dependent leukemia cell survival. Chem. Biol. 19, 1175–1186 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Bird, G. H., Bernal, F., Pitter, K. & Walensky, L. D. Synthesis and biophysical characterization of stabilized alpha-helices of BCL-2 domains. Methods Enzymol. 446, 369–386 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Chen, Y. H., Yang, J. T. & Chau, K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry 13, 3350–3359 (1974).

    CAS  PubMed  Article  Google Scholar 

  51. Lamiable, A. et al. PEP-FOLD3: faster de novo structure prediction for linear peptides in solution and in complex. Nucleic Acids Res. 44, W449–W454 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Shen, Y., Maupetit, J., Derreumaux, P. & Tuffery, P. Improved PEP-FOLD approach for peptide and miniprotein structure prediction. J. Chem. Theory Comput. 10, 4745–4758 (2014).

    CAS  PubMed  Article  Google Scholar 

  53. Thevenet, P. et al. PEP-FOLD: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides. Nucleic Acids Res. 40, W288–W293 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Barclay, L. A. et al. Inhibition of pro-apoptotic BAX by a noncanonical interaction mechanism. Mol. Cell 57, 873–886 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2019).

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

We thank E. Smith for graphics support and assistance with figure preparation, and D.T.C. for helpful discussions. The study was funded in part by NIH grant nos. T32GM007753, T32GM008313 and 5F30CA221087 to A.M.C.; NIH grant no. R50CA211399 to G.H.B.; Lauri Strauss Leukemia Foundation Discover grant to H.D.H.; NIH grant no. 5F31CA210592 to E.P.H.; NIH grant no. 5F31CA210590 to Z.J.H.; NIH grant no. T32GM007753 to C.E.N.; Landry Cancer Biology Research Fellowship and Chleck Family Scholarship to U.A.; NSF predoctoral fellowship to M.S.P.; NIH grant no. R01GM101135 to J.R.E. and NIH grant no. R35CA197583 to L.D.W. Additional support was provided by a research collaboration between J.R.E. and the Waters Corporation. We also thank the Wolpoff Family Foundation, J. and L. LaTorre, the family of I. Coll and the Todd J. Schwartz Memorial Fund for their financial contributions to our cancer chemical biology research.

Author information

Authors and Affiliations

Authors

Contributions

A.M.C. and L.D.W. designed the study. G.H.B. and T.O. synthesized, purified and characterized the stapled peptides, and performed circular dichroism and peptide proteolysis assays. A.M.C. conducted all of the biochemical studies, except for the VLCAD enzyme assay and ubiquitin pathway reconstitution assays performed by M.S.P. and H.D.H., respectively. H.D.H. performed the fluorescent bead binding assay, and U.A. contributed to peptide specificity of action assays. A.M.C., T.E.W., S.L., E.P.H., Z.J.H. and C.E.N. performed and analyzed the HXMS analyses under the supervision of J.R.E. L.D.W. and A.M.C. wrote the manuscript, which was reviewed by all co-authors.

Corresponding author

Correspondence to Loren D. Walensky.

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L.D.W. is a scientific co-founder and shareholder in Aileron Therapeutics.

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Extended data

Extended Data Fig. 1 Circular dichroism analysis of Ubc15-1 and SAH-Ubc15-11.

(a-b) Circular dichroism spectra of Ubc15-1 (BPSSASRQLLRKQLKEIQ) (a) and SAH-Ubc15-11 (b), demonstrating induction of α-helicity by insertion of the i, i+7 staple at positions S6 and Q13. B, norleucine (replacement for methionine, whose sulfur residue decreases the efficiency of ruthenium-catalyzed olefin metathesis). Source data for CD spectra are available online.

Source data

Extended Data Fig. 2 Specificity of action of SAH-UBE2A.

a, Dose-responsive inhibition of UBE1 by SAH-UBE2A in the absence (Fig. 2b) or presence of 0.01% Triton X-100, as monitored by thioester transfer assay, gel electrophoresis, and silver stain. b, Dose-responsive inhibition of UBE1 by SAH-UBE2A using reaction buffer containing 50 mM (standard; Fig. 2b) or 250 mM (elevated) NaCl, as monitored by thioester transfer assay, gel electrophoresis, and silver stain. c, Fluorescent bead binding assay showing that FITC-SAH-UBE2A exhibits no non-specific binding to Ni-NTA beads (top row), selective binding to His-UBE1 coated beads (middle row), and no non-specific binding to beads coated with a negative control protein, such as His-p53 (bottom row). TLM, transmission light microscopy. Scale bars, 200 μm. d, VLCAD enzymatic assay performed in the presence of vehicle or SAH-UBE2A (30:1 peptide:enzyme ratio, final peptide concentration 43.2 μM), demonstrating no interference by SAH-UBE2A with the capacity of VLCAD to oxidize its palmitoyl-CoA substrate. Data are mean ± s.e.m. for experiments performed in technical triplicate. e, In vitro ubiquitin pathway reconstitution assay showing that SAH-UBE2A (30 μM, excess) inhibits ubiquitination of p53 when the cascade relies on E1 thioester transfer activity but not when the E1 step is bypassed by the addition of E2 pre-charged with ubiquitin. In addition, SAH-UBE2A has no effect on the capacity of the DUB USP7 to deubiquitinate p53. The entire experiment was performed twice using independent preparations of reagents with similar results. f, SAH-UBE2A has no effect on the capacity of UBE1 to form a covalent UBE1~ubiquitin thioester adduct, the enzymatic step of the UBE1 catalytic cycle that precedes thioester transfer to E2, as demonstrated by the presence of a UBE1 and UBE1~Ub doublet (silver stain) across all SAH-UBE2A concentrations of the thioester transfer assay. g, To verify that the E1-inhibitory mechanism was not dependent on ubiquitin discharge from E2 onto SAH-UBE2A, a lysine-free K14R mutant was synthesized and tested in the thioester transfer assay. Both SAH-UBE2A and SAH-UBE2AK14R (10 μM dosing) showed equivalent E1-inhibitory activity, as reflected by blockade of E2~Ub formation. Uncropped gels for panels a, b, e-f are available online. Source data for the VLCAD enzymatic assay plot are available online.

Source data

Extended Data Fig. 3 SAH-UBE2A binds to UBE1 in the presence and absence of ubiquitin and ATP.

a, Similar protection of SAH-UBE2A from deuterium exchange upon incubation with UBE1 (2:1 protein:peptide) in the presence (black) or absence (light gray) of ubiquitin and ATP. The relative difference in deuterium uptake after 10 s of labeling is shown. The difference in uptake was calculated from the mean deuterium level for SAH-UBE2A in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. b, C-terminally FITCylated SAH-UBE2A bound to UBE1 with respective Kds of 384 ± 43 nM and 359 ± 60 nM in the absence (light gray) and presence (black) of ubiquitin and ATP, as assessed by fluorescence polarization assay. Kds were determined by nonlinear regression analysis of experiments performed in technical triplicate (with ubiquitin/ATP) and plotted alongside the data shown in Fig. 2i (without ubiquitin/ATP). The two curve fits have R2 values of 0.9491 (without ubiquitin/ATP) and 0.9636 (with ubiquitin/ATP). Source data for the HXMS and FP plots are available online.

Source data

Extended Data Fig. 4 Circular dichroism analysis of SAH-UBE2A and its R-to-E point mutants.

Circular dichroism spectra of SAH-UBE2A and its R6E, R7E, and R8E single point mutants, showing a similar degree of high α-helicity for the R-to-E mutants. Source data for the CD spectra are available online.

Source data

Extended Data Fig. 5 Deuterium exchange profile of UBE1.

a, The hydrogen-deuterium exchange profile of UBE1 was measured at 10 s, 1 min, and 10 min of deuterium labeling and the average deuterium uptake (numerical values are found in Supplementary Data File 1) over time for three independent biological replicates displayed with a chiclet plot (see also Supplementary Table 1). Exemplary regions of early uptake are labeled with red arrows and text, whereas exemplary areas that show gradual uptake over time are labeled with black arrows and text. The structurally-defined regions of E1, including the SCCH, FCCH, IAD, AAD, and UFD, are shown in the key to the right of the chiclet plot. b, The regions and extent of deuterium uptake are mapped onto the structure of E1 (PDB: 6DC6) in accordance with the color scale shown. Regions of high deuterium uptake at the earliest 10 s time point, reflective of solvent exposure, include, for example, the N-terminal extension, cysteine cap, and crossover loop. Regions that demonstrate increasing exchange over time, consistent with dynamic structures, include N-terminal alpha-helices, adenylation active site, SCCH (excluding the cysteine cap), re-entry loop, and UFD linker hinge. Conformationally stable regions that show no deuterium uptake (light gray) at any of the time points include α-helices and beta sheets of the IAD and AAD protein core.

Extended Data Fig. 6 Single R7E and triple R7A/L9A/B10A mutagenesis progressively inhibit the functional and conformational effects observed upon SAH-UBE2A/UBE1 interaction.

a, Sequence compositions of the unmodified UBE2A template peptide and stapled constructs SAH-UBE2A, SAH-UBE2AR7E, and SAH-UBE2AR7A/L9A/B10A. b, Comparative effects of UBE2A, SAH-UBE2A, SAH-UBE2AR7E, and SAH-UBE2AR7A/L9A/B10A on UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2. Whereas SAH-UBE2A (10 μM) completely inhibits UBE1 activity, triple mutagenesis abrogates the inhibitory effect and single R7E mutagenesis has an intermediate effect. c, Progressive loss of UBE1 protection from deuterium exchange into SAH-UBE2A upon single R7E and triple R7A/L9A/B10A mutagenesis. The relative difference in deuterium uptake after 10 s of labeling is shown. The difference in uptake was calculated from the mean deuterium level for each SAH-UBE2A peptide in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. d, Difference in deuterium uptake plots demonstrate the relative deuterium incorporation of SAH-UBE2A/UBE1, SAH-UBE2AR7E/UBE1, or SAH-UBE2AR7A/L9A/B10A/UBE1 minus the relative deuterium incorporation of UBE1 at 10 min of deuterium labeling. The shaded gray region indicates differences in deuteration that are below the meaningful differences threshold. Consistent with results of both the thioester transfer assay (b) and peptide deuterium exchange (c), and the predicted SAH-UBE2A/UBE1 binding mode, triple mutagenesis essentially abrogates the effect of SAH-UBE2A on UBE1 conformation, with single R7E mutagenesis having an intermediate influence. Data are representative of three independent replicates for SAH-UBE2A, and two independent replicates for SAH-UBE2AR7E and SAH-UBE2AR7A/L9A/B10A. Each peptide, from N- to C-terminus, was given a peptide number to simplify the presentation. The peptide list and residue identity of each peptide can be found in Supplementary Data File 1. Uncropped gel for panel b is available online. Source data for the HXMS analyses are available online.

Source data

Extended Data Fig. 7 E2 proteins compete with SAH-UBE2A for UBE1 interaction.

Fluorescent bead binding assay showing that the binding of FITC-SAH-UBE2A (0.5 μM) to His-UBE1 coated Ni-NTA beads (top row) is blocked by preincubation of the UBE-1 beads with excess UBE2A, (second row), UBE2D1 (third row), and UBE2T (fourth row) proteins (25 μM). Scale bars, 200 μm.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Tables 1 and 2.

Reporting Summary

Supplementary Data

HXMS data for analyses of UBE1 and the UBE1/SAH-UBE2A interactions.

Source data

Source Data Fig. 1

Uncropped thioester transfer assay gel.

Source Data Fig. 1

Data for thioester transfer assays.

Source Data Fig. 2

Uncropped thioester transfer assay gels and pulldown gel.

Source Data Fig. 2

Data for thioester transfer assays, CD spectra, protease resistance testing, HXMS analyses and FP plot.

Source Data Fig. 3

Uncropped thioester transfer assay gels.

Source Data Fig. 3

Data for thioester transfer assays.

Source Data Fig. 5

Uncropped thioester transfer assay gels.

Source Data Fig. 5

Data for thioester transfer assays.

Source Data Extended Data Fig. 1

Data for CD spectra.

Source Data Extended Data Fig. 2

Uncropped thioester transfer assay gels, p53 ubiquitination blot and E1-ubiquitination gel.

Source Data Extended Data Fig. 2

Data for VLCAD enzymatic assay.

Source Data Extended Data Fig. 3

Data for HXMS and FP plots.

Source Data Extended Data Fig. 4

Data for CD spectra.

Source Data Extended Data Fig. 6

Uncropped thioester transfer assay gel.

Source Data Extended Data Fig. 6

Data for HXMS analyses.

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Cathcart, A.M., Bird, G.H., Wales, T.E. et al. Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer. Nat Chem Biol 16, 1218–1226 (2020). https://doi.org/10.1038/s41589-020-0625-7

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