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Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms

Nature Chemistry (2024)Cite this article

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Abstract

Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein semisynthesis, chemical rescue, microscale thermophoresis and other biochemical approaches to dissect the role of catalytic base/acid function and conformational interconversion in HECT-domain E3 catalysis. We demonstrate that there is plasticity in the use of the terminal side chain or backbone carboxylate for proton transfer in HECT E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation.

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Fig. 1: Enzymatic analysis of NEDD4 C-terminal Asp mutants.
Fig. 2: Chemical rescue of NEDD4 mutants.
Fig. 3: Analysis of the C-terminal backbone carboxylate in HECT enzymes.
Fig. 4: Versatility of carboxylate usage in Rsp5 E3 ligase.
Fig. 5: Analysis of protein–protein interaction using ubiquitination intermediates (E3-Ub or E2-Ub).
Fig. 6: Evolutionary analysis of HECT E3 ligases.

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Data availability

The protein MS raw data, Rosetta modelling raw file, AlphaFold raw file, ML phylogeny analysis and all raw uncropped image files for gels and western blots of this study have been deposited at Harvard Dataverse (https://doi.org/10.7910/DVN/BFMQU6). Source data are provided with this paper.

Code availability

The source codes for Rosetta modelling of the NEDD4-1 C-terminal tail, AlphaFold2 modelling of Rsp5-Ub-WBP2 and the HECT ML phylogenetic analysis are accessible in Supplementary Codes 1, 2 and 3.

References

  1. Hershko, A. & Ciechanover, A. The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61, 761–807 (1992).

    Article  CAS  PubMed  Google Scholar 

  2. Scheffner, M., Nuber, U. & Huibregtse, J. M. Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade. Nature 373, 81–83 (1995).

    Article  CAS  PubMed  Google Scholar 

  3. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Swatek, K. N. & Komander, D. Ubiquitin modifications. Cell Res. 26, 399–422 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Harper, J. W. & Schulman, B. A. Cullin-RING ubiquitin ligase regulatory circuits: a quarter century beyond the F-box hypothesis. Annu. Rev. Biochem. 90, 403–429 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rotin, D. & Kumar, S. Physiological functions of the HECT family of ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 10, 398–409 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Zheng, N. & Shabek, N. Ubiquitin ligases: structure, function and regulation. Annu. Rev. Biochem. 86, 129–157 (2017).

    Article  CAS  PubMed  Google Scholar 

  8. Weber, J., Polo, S. & Maspero, E. HECT E3 ligases: a tale with multiple facets. Front. Physiol. 10, 370 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Sakamoto, K. M. et al. Protacs: chimeric molecules that target proteins to the Skp1–Cullin–F box complex for ubiquitination and degradation. Proc. Natl Acad. Sci. USA 98, 8554–8559 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fischer, E. S. et al. Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Békés, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nat. Rev. Drug Discov. 21, 181–200 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Huibregtse, J. M., Scheffner, M., Beaudenon, S. & Howley, P. M. A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl Acad. Sci. USA 92, 2563–2567 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bernassola, F., Karin, M., Ciechanover, A. & Melino, G. The HECT family of E3 ubiquitin ligases: multiple players in cancer development. Cancer Cell 14, 10–21 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Maddika, S. et al. WWP2 is an E3 ubiquitin ligase for PTEN. Nat. Cell Biol. 13, 728–733 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Scheffner, M. & Kumar, S. Mammalian HECT ubiquitin-protein ligases: biological and pathophysiological aspects. Biochim. Biophys. Acta 1843, 61–74 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. Buetow, L. & Huang, D. T. Structural insights into the catalysis and regulation of E3 ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 17, 626–642 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J. & Howley, P. M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 1129–1136 (1990).

    Article  CAS  PubMed  Google Scholar 

  18. Scheffner, M., Huibregtse, J. M., Vierstra, R. D. & Howley, P. M. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75, 495–505 (1993).

    Article  CAS  PubMed  Google Scholar 

  19. Trotman, L. C. et al. Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128, 141–156 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang, X. et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 128, 129–139 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Jiang, H. et al. Enzymatic analysis of WWP2 E3 ubiquitin ligase using protein microarrays identifies autophagy-related substrates. J. Biol. Chem. 298, 101854 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fajner, V., Maspero, E. & Polo, S. Targeting HECT-type E3 ligases—insights from catalysis, regulation and inhibitors. FEBS Lett. 591, 2636–2647 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Lorenz, S. Structural mechanisms of HECT-type ubiquitin ligases. Biol. Chem. 399, 127–145 (2018).

    Article  CAS  PubMed  Google Scholar 

  24. Verdecia, M. A. et al. Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase. Mol. Cell 11, 249–259 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Maspero, E. et al. Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming. Nat. Struct. Mol. Biol. 20, 696–701 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Paik, W. K., Pearson, D., Lee, H. W. & Kim, S. Nonenzymatic acetylation of histones with acetyl-CoA. Biochim. Biophys. Acta 213, 513–522 (1970).

    Article  CAS  PubMed  Google Scholar 

  27. Berg, J. M. & Lorsch, J. R. Mechanism of ribosomal peptide bond formation. Science 291, 203 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Wolf, E., De Angelis, J., Khalil, E. M., Cole, P. A. & Burley, S. K. X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition. J. Mol. Biol. 317, 215–224 (2002).

    Article  CAS  PubMed  Google Scholar 

  29. Kee, Y. & Huibregtse, J. M. Regulation of catalytic activities of HECT ubiquitin ligases. Biochem. Biophys. Res. Commun. 354, 329–333 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jencks, W. P. Requirements for general acid-base catalysis of complex reactions. J. Am. Chem. Soc. 94, 4731–4732 (1972).

    Article  CAS  Google Scholar 

  31. Richard, J. P., Huber, R. E., Heo, C., Amyes, T. L. & Lin, S. Structure-reactivity relationships for β-galactosidase (Escherichia coli, lac Z). 4. Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermediates of E461G and E461Q β-galactosidases. Biochemistry 35, 12387–12401 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. Huang, L. et al. Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science 286, 1321–1326 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Kamadurai, H. B. et al. Insights into ubiquitin transfer cascades from a structure of a UbcH5B approximately ubiquitin-HECT(NEDD4L) complex. Mol. Cell 36, 1095–1102 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kim, H. C., Steffen, A. M., Oldham, M. L., Chen, J. & Huibregtse, J. M. Structure and function of a HECT domain ubiquitin-binding site. EMBO Rep. 12, 334–341 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kamadurai, H. B. et al. Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3. eLife 2, e00828 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Nair, R. M. et al. Reconstitution and structural analysis of a HECT ligase-ubiquitin complex via an activity-based probe. ACS Chem. Biol. 16, 1615–1621 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hunkeler, M. et al. Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition. Mol. Cell 81, 3468–3480 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Grabarczyk, D. B. et al. HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain. Nat. Chem. Biol. 17, 1084–1092 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hehl, L. A. et al. Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5. Nat. Chem. Biol. 20, 190–200 (2024).

    Article  CAS  PubMed  Google Scholar 

  40. Mao, J. et al. Structural visualization of HECT-E3 Ufd4 accepting and transferring ubiquitin to form K29/K48-branched polyubiquitination on N-degron. Preprint at bioRxiv https://www.biorxiv.org/content/10.1101/2023.05.23.542033v1 (2023).

  41. Tsai, J. M. et al. UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability. Mol. Cell 83, 2753–2767 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mark, K. G. et al. Orphan quality control shapes network dynamics and gene expression. Cell 186, 3460–3475 (2023).

    Article  CAS  PubMed  Google Scholar 

  43. Dring, J. et al. Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1. Nat. Struct. Mol. Biol. 31, 364–377 (2024).

    Article  Google Scholar 

  44. Wiesner, S. et al. Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Cell 130, 651–662 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Jiang, H., Thomas, S. N., Chen, Z., Chiang, C. Y. & Cole, P. A. Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases. J. Biol. Chem. 294, 17421–17436 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl Acad. Sci. USA 95, 6705–6710 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chu, N. et al. Akt kinase activation mechanisms revealed using protein semisynthesis. Cell 174, 897–907 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Cole, P. A. et al. Chemical approaches to reversible protein phosphorylation. Acc. Chem. Res. 36, 444–452 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Qiao, Y., Molina, H., Pandey, A., Zhang, J. & Cole, P. A. Chemical rescue of a mutant enzyme in living cells. Science 311, 1293–1297 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Maupin, C. M. et al. Chemical rescue of enzymes: proton transfer in mutants of human carbonic anhydrase II. J. Am. Chem. Soc. 133, 6223–6234 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Jencks, W. P. Catalysis in Chemistry and Enzymology (Courier Corporation, 1987).

  52. Kim, H. C. & Huibregtse, J. M. Polyubiquitination by HECT E3s and the determinants of chain type specificity. Mol. Cell. Biol. 29, 3307–3318 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kathman, S. G. et al. A small molecule that switches a ubiquitin ligase from a processive to a distributive enzymatic mechanism. J. Am. Chem. Soc. 137, 12442–12445 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Schowen, K. B. & Schowen, R. L. Solvent isotope effects on enzyme systems. Methods Enzymol. 87, 551–606 (1982).

    Article  CAS  PubMed  Google Scholar 

  55. Bhardwaj, G. et al. Accurate de novo design of hyperstable constrained peptides. Nature 538, 329–335 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Evans, R. et al. Protein complex prediction with AlphaFold-Multimer. Preprint at bioRxiv https://www.biorxiv.org/content/10.1101/2021.10.04.463034v1 (2021).

  57. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Bhat, S. et al. Hydrazide mimics for protein lysine acylation to assess nucleosome dynamics and deubiquitinase action. J. Am. Chem. Soc. 140, 9478–9485 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Dorr, B. M., Ham, H. O., An, C., Chaikof, E. L. & Liu, D. R. Reprogramming the specificity of sortase enzymes. Proc. Natl Acad. Sci. USA 111, 13343–13348 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Jiang, H. & Cole, P. A. N-terminal protein labeling with N-hydroxysuccinimide esters and microscale thermophoresis measurements of protein-protein interactions using labeled protein. Curr. Protoc. 1, e14 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Zhang, W. et al. System-wide modulation of HECT E3 ligases with selective ubiquitin variant probes. Mol. Cell 62, 121–136 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Mortensen, F. et al. Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination. Proc. Natl Acad. Sci. USA 112, 9872–9877 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Salvat, C., Wang, G., Dastur, A., Lyon, N. & Huibregtse, J. M. The −4 phenylalanine is required for substrate ubiquitination catalyzed by HECT ubiquitin ligases. J. Biol. Chem. 279, 18935–18943 (2004).

    Article  CAS  PubMed  Google Scholar 

  64. Jackl, M. et al. β-sheet augmentation is a conserved mechanism of priming HECT E3 ligases for ubiquitin ligation. J. Mol. Biol. 430, 3218–3233 (2018).

    Article  PubMed  Google Scholar 

  65. Eletr, Z. M., Huang, D. T., Duda, D. M., Schulman, B. A. & Kuhlman, B. E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nat. Struct. Mol. Biol. 12, 933–934 (2005).

    Article  CAS  PubMed  Google Scholar 

  66. Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems (Wiley, 1975).

  67. Ruben, E. A. et al. Ground state destabilization from a positioned general base in the ketosteroid isomerase active site. Biochemistry 52, 1074–1081 (2013).

    Article  CAS  PubMed  Google Scholar 

  68. Liwocha, J. et al. Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler. Nat. Chem. Biol. 17, 272–279 (2021).

    Article  CAS  PubMed  Google Scholar 

  69. Liu, M. Y., Gutowski, S. & Sternweis, P. C. The C terminus of mammalian phospholipase D is required for catalytic activity. J. Biol. Chem. 276, 5556–5562 (2001).

    Article  CAS  PubMed  Google Scholar 

  70. Farazi, T. A., Manchester, J. K., Waksman, G. & Gordon, J. I. Pre-steady-state kinetic studies of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase mutants identify residues involved in catalysis. Biochemistry 40, 9177–9186 (2001).

    Article  CAS  PubMed  Google Scholar 

  71. Koh, C. S., Madireddy, R., Beane, T. J., Zamore, P. D. & Korostelev, A. A. Small methyltransferase RlmH assembles a composite active site to methylate a ribosomal pseudouridine. Sci. Rep. 7, 969 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Grau-Bové, X., Sebé-Pedrós, A. & Ruiz-Trillo, I. A genomic survey of HECT ubiquitin ligases in eukaryotes reveals independent expansions of the HECT system in several lineages. Genome Biol. Evol. 5, 833–847 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Fang, N. N. et al. Rsp5/Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress. Nat. Cell Biol. 16, 1227–1237 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Riling, C. et al. Itch WW domains inhibit its E3 ubiquitin ligase activity by blocking E2-E3 ligase trans-thiolation. J. Biol. Chem. 290, 23875–23887 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Chen, Z. et al. A tunable brake for HECT ubiquitin ligases. Mol. Cell 66, 345–357 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Zhu, K. et al. Allosteric auto-inhibition and activation of the Nedd4 family E3 ligase Itch. EMBO Rep 18, 1618–1630 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Wang, Z. et al. A multi-lock inhibitory mechanism for fine-tuning enzyme activities of the HECT family E3 ligases. Nat. Commun. 10, 3162 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Conway, P., Tyka, M. D., DiMaio, F., Konerding, D. E. & Baker, D. Relaxation of backbone bond geometry improves protein energy landscape modeling. Protein Sci. 23, 47–55 (2014).

    Article  CAS  PubMed  Google Scholar 

  79. Lee, K., Whedon, S. D., Wang, Z. A. & Cole, P. A. Distinct biochemical properties of the class I histone deacetylase complexes. Curr. Opin. Chem. Biol. 70, 102179 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Elfmann, C. & Stülke, J. PAE viewer: a webserver for the interactive visualization of the predicted aligned error for multimer structure predictions and crosslinks. Nucleic Acids Res. 51, W404–W410 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Nakamura, T., Yamada, K. D., Tomii, K. & Katoh, K. Parallelization of MAFFT for large-scale multiple sequence alignments. Bioinformatics 34, 2490–2492 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Rozewicki, J., Li, S., Amada, K. M., Standley, D. M. & Katoh, K. MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Res. 47, W5–W10 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Kozlov, A. M., Darriba, D., Flouri, T., Morel, B. & Stamatakis, A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 35, 4453–4455 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Miller, M. A., Pfeiffer, W. & Schwartz, T. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In Proc. 2010 gateway computing environments workshop (GCE) 1–8 (IEEE, 2010).

  85. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Derelle, R. & Lang, B. F. Rooting the eukaryotic tree with mitochondrial and bacterial proteins. Mol. Biol. Evol. 29, 1277–1289 (2012).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work is supported by the National Institutes of Health (grants nos. R01GM62437 and R01CA74305 to P.A.C.), the Claudia Adams Barr Program for Innovative Cancer Research (to H.A. and P.A.C.), the Loeb Center Fellowship (to H.K.) and an American Heart Association Postdoctoral fellowship (grant no. 826614 to K.L.). We thank J. Lee at Dana Farber Cancer Institute for assistance with MS and K. Arnett at Harvard CMI core for help with MST experiments. We thank J. P. Richard and Z. Chen for their helpful comments on this manuscript. We are grateful to S. Sunyaev for helpful discussions on the evolution analysis. We also appreciate the help from the Research Computing Group at Harvard Medical School for providing computing resources. We are grateful for the helpful advice from Cole laboratory members, and we thank C. Wolberger at Johns Hopkins University, W. Wei at Beth Israel Deaconess Medical Center, W. Harper at Harvard Medical School and X. Jiang at Sloan Kettering Institute for sharing reagents.

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Authors and Affiliations

  1. Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA

    Hanjie Jiang, Bryant D. Miller, Hyojeon Kim, Kwangwoon Lee & Philip A. Cole

  2. Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Hanjie Jiang, Thibault Viennet, Hyojeon Kim, Kwangwoon Lee, Haribabu Arthanari & Philip A. Cole

  3. Department of Human Biology, Sattler College, Boston, MA, USA

    Bryant D. Miller

  4. Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA

    Thibault Viennet & Haribabu Arthanari

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Contributions

Conceptualization and experimental design were carried out by H.J., B.D.M., T.V., H.K., K.L., H.A. and P.A.C. Investigations were performed by H.J., B.D.M., T.V. and H.K. Data analysis was carried out by H.J., B.D.M., T.V., H.K., K.L., H.A. and P.A.C. The original draft was written by H.J., T.V., K.L., H.A. and P.A.C. Review and editing were performed by H.J., B.D.M., T.V., H.K., K.L., H.A. and P.A.C.

Corresponding author

Correspondence to Philip A. Cole.

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All collaborators of this study have fulfilled the criteria for authorship required by Nature Portfolio journals have been included as authors, as their participation was essential for the design and implementation of the Study. Roles and responsibilities were agreed among collaborators ahead of the research. This research was not severely restricted or prohibited in the setting of the researchers, and does not result in stigmatization, incrimination, discrimination or personal risk to participants. Local and regional research relevant to our study was taken into account in citations.

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Extended data

Extended Data Fig. 1 Analyses of mutant recombinant and semisynthetic NEDD4 and NEDD4L.

(a) In vitro ubiquitination assays comparing activity of recombinantly expressed NEDD4L protein with D955N or D955A mutations (P6-P8). The unmodified NEDD4L band was quantified by densitometry. The percentage of unmodified NEDD4L species versus time zero in each case is listed below the lanes (n = 3 biologically independent experiments showed similar results). (b) In vitro ubiquitination assay comparing the enzyme activity of recombinantly expressed NEDD4 proteins including WT (P1), ΔD897/D900E (P9), D900E (P2), and D900A (P4). The unmodified NEDD4 band was quantified by densitometry. The percentage of unmodified NEDD4 species in each case is listed below the lanes (n = 2 biologically independent experiments showed similar results). (c) In vitro ubiquitination assays comparing enzyme activity of recombinantly expressed NEDD4 WT (P1), T893C (P10), with semisynthetic NEDD4 with C-terminal carboxylate (P11). The unmodified NEDD4 band was quantified by densitometry and the percentage of unmodified NEDD4 species is listed below the lanes (n = 2 biologically independent experiments showed similar results). (d) LC-MS spectrum of NEDD4 D900-phospho-serine (P12) made by expressed protein ligation. (e) LC-MS spectrum of NEDD4 D900-homo-cysteine (P13) made by expressed protein ligation. (f) LC-MS spectrum of NEDD4 D900-sulfonyl-alanine (P14) made by expressed protein ligation. (g) LC-MS spectrum of NEDD4 D900-d-Asp (P15) made by expressed protein ligation. (h) LC-MS spectrum of NEDD4 D900-d-Glu (P16) made by expressed protein ligation. (i) In vitro ubiquitination assay comparing the activity of NEDD4 D900 substitutions to d-Asp (dD, P15) or d-Glu (dE, P16). The unmodified NEDD4 bands were quantified by densitometry. The percentages of unmodified NEDD4 species versus time zero are listed below the graph (n = 2 biologically independent experiments showed similar results).

Source data

Extended Data Fig. 2 Chemical rescue of NEDD4 mutant.

(a) Chemical rescue assays of NEDD4 D900A (P4) with acetate, difluoroacetate (DFA, pKa 1.3) and trifluoroacetate (TFA). The α-ubiquitin western blot signal from the highest concentration of each reagent was used for densitometry quantification and plotted relative to the acetate-rescued NEDD4 D900A activity in the bar graph (n = 3 biologically independent experiments showed similar results). Data are presented as mean values ± SEM. For the catalytically defective D900A NEDD4-1, the assays were conducted at 37 °C with 5 μM E2 to enhance the baseline activities. The same reaction conditions were used for other chemical rescue assays below. (b) Chemical rescue assays of NEDD4 D900A (P4) with acetate, fluoroacetate (FA, pKa 2.6) and trifluoroacetate (TFA). Results were analyzed by densitometry and plotted relative to the acetate-rescued NEDD4 D900A activity in the bar graph (n = 3 biologically independent experiments showed similar results). Data are presented as mean values ± SEM. (c) Chemical rescue assays of NEDD4 D900A (P4) with formate (pKa 3.8), acetate and propionate (pKa 4.9). Results were analyzed by densitometry and plotted relative to the acetate- rescued NEDD4 D900A activity in the bar graph (n = 3 biologically independent experiments showed similar results). Data are presented as mean values ± SEM. (d) Chemical rescue assays of NEDD4 D900A (P4) with acetate and phosphate (pKa 6.8). Results were analyzed by densitometry and plotted to reflect the relative activity in the bar graph (n = 3 biologically independent experiments showed similar results). Data are presented as mean values ± SEM. (e) Chemical rescue assay of NEDD4 D900A (P4) with acetate in deuterated water (2H2O). Autoubiquitination was analyzed by densitometry using the highest concentration of acetate in 2H2O. The quantification was plotted to compare the relative activity of NEDD4 in the absence and presence of acetate under 2H2O (n = 4 biologically independent experiments showed similar results). Data are presented as mean values ± SEM.

Source data

Extended Data Fig. 3 Characterization of semisynthetic NEDD4 and sequence comparison among NEDD4, HUWE1 and E6AP proteins.

(a) LC-MS spectrum of NEDD4 D900-carboxamide (P18). (b) LC-MS spectrum of NEDD4 ΔD900 V899-carboxylate (P19). (c) LC-MS spectrum of NEDD4 ΔD900 V899-carboxamide (P20). (d) LC-MS spectrum of NEDD4 ΔD900 V899-βVal (P21). (e) Sequence alignment of human NEDD4, HUWE1 and E6AP. The C-terminal residues are highlighted in the red box. Homologous residues of NEDD4 (V588) and HUWE1 (L4061) are highlighted with a red arrow.

Extended Data Fig. 4 Characterization of the C-terminal residue in HUWE1, E6AP, WWP2 and Rsp5 protein catalysis.

(a) Semisynthetic strategy to make HUWE1 HECT proteins containing different C-termini. (b) Semisynthetic strategy to make E6AP proteins containing different C-termini. (c) LC-MS spectrum of the HUWE1 HECT domain protein containing an A4374-carboxylate (P23) made by expressed protein ligation. (d) LC-MS spectrum of the HUWE1 HECT domain protein containing an A4374-carboxamide (P24) made by expressed protein ligation. (e) Ubiquitination assays of E6AP protein with UbcH5b E2 protein. Semisynthetic E6AP proteins containing carboxylate or carboxamide C termini (P25 and P26). The assays were conducted at 37 °C with 5 μM E2. Quantification of ubiquitination smear western blot signals was used to compare the relative activities (n = 3 biologically independent experiments showed similar results). Data are presented as mean values ± SEM. (f) In vitro ubiquitination assays comparing activity of recombinantly expressed WWP2 protein with E870Q or E870A mutations (P33-P35). The unmodified WWP2 band was quantified by densitometry and the percentage of unmodified species is listed below the lanes (n = 3 biologically independent experiments showed similar results). (g) Representative AlphaFold2 multimer structure of the Rsp5-Ub-WBP2 complex (without Rosetta refinement). Three ubiquitins were used to capture the Rsp5 in the L-shape containing Ub in the active site. Two ubiquitin molecules engage the allosteric ubiquitin-binding site (Ub1) and E2 binding site (Ub2) on the N-lobe, whereas the third ubiquitin (Ub3) sits in the E3 active site to mimic the Ub-loaded state, reminiscent of the previously reported Ub-loaded Rsp5 crystal structure trapped in the L-shape (PDB: 4LCD). (h) The predicted aligned error (PAE) plot for the AlphaFold2 structure of the Rsp5-Ub-WBP2 complex shown in panel G. (i) Superimposed structures of the Rsp5-Ub-WBP2 complex from in silico modeling with the Rsp5-Ub-Sna3 crystal structure (PDB: 4LCD). Despite differences in the bound substrates (WBP2 versus Sna3) and rounds of structural refinements (for Rsp5-Ub-WBP2), the two structures align reasonably well (RMSD of 1.113 Å). The recognition of different substrates likely causes orientational differences in the flexible WW3 domain between the two structures, as WW3 provides a principal substrate binding platform for proline-rich motif in substrates.

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Extended Data Fig. 5 MST analysis of E3/E3-Ub binding to E2 or substrate.

(a) In vitro ubiquitination comparing the relative activity of NEDD4 WT versus the single Cys form of NEDD4. The single Cys form contains three Cys to Ser replacements. The percentage of unmodified NEDD4 species in each case is listed below the lanes (n = 2 biologically independent experiments showed similar results). (b) In vitro ubiquitination comparing the relative activity of WWP2 WT versus single Cys form of WWP2. The single Cys form of WWP2 contains seven Cys to Ser/Ala replacements. The percentage of unmodified WWP2 species in each case is listed below the lanes. The single Cys WWP2 demonstrates robust activity, more active than the WT, which might result from loosened linker autoinhibition (n = 2 biologically independent experiments showed similar results). (c) Chemical details of NEDD4 Cys ubiquitin linkages. The native thioester linkage, vinyl thioether linkage (made with a Ub propargyl probe), and hydrazide mimic linkage (made using the Ub hydrazide method) are compared. (d) MST analysis of UbcH5b E2 protein binding with NEDD4-Ub prepared with a Ub-Prg activity-based probe to form a vinyl-thioester linkage (P41). The MST bound fractions as a function of E2 protein concentrations are shown. The equilibrium dissociation constant Kd values were calculated from three repeats using a quadratic binding model (n = 3 biologically independent experiments). (e) MST analysis of UbcH5b E2 protein binding with NEDD4-Ub prepared with Ub-hydrazide with the last Ub Gly76 deleted (P42) to approximate the linker length of a native Ub-Cys thioester linkage (n = 3 biologically independent experiments). (f) MST analysis of UbcH7 E2 binding with WWP2 or WWP-Ub (P39 and P40) (n = 2 biologically independent experiments). (g) MST analysis of UbcH5b E2 binding with NEDD4 HECT or HECT-Ub (P44 and P45). The Kd values were calculated from repeats using a quadratic binding model (n = 2 biologically independent experiments). (h) MST analysis of UbcH5b E2 binding with NEDD4 HECT or HECT-Ub (P44 and P45) in the presence of WT Ub at 100 μM (n = 2 biologically independent experiments). (i) MST analysis of UbcH5b E2 binding with NEDD4 HECT or HECT-Ub (P44 and P45) in the presence of UbV (5 μM) (n = 2 biologically independent experiments). (j) Fluorescence anisotropy analysis of UbV binding to NEDD4 or NEDD4-Ub (n = 2 biologically independent experiments). (k) MST analysis of substrate NDP52 (P46) binding to NEDD4 (P36) or NEDD4-Ub (P37). The Kd values were calculated from two repeats using a quadratic binding model (n = 2 biologically independent experiments). (l) MST analysis of substrate WBP2 binding with NEDD4 or NEDD4-Ub (P36 and P37). The Kd values were calculated from repeats using a quadratic binding model (n = 2 biologically independent experiments). (m) Fluorescence anisotropy analysis of NEDD4 or NEDD4-Ub protein binding to E2 (UbcH5b). The E2 protein is labeled with fluorescein (P50) and the Kd was calculated using a quadratic binding model (n = 2 biologically independent experiments). (n) Fluorescence anisotropy analysis of NEDD4 protein binding to E2 (UbcH5b)-Ub. The E2-Ub protein is labeled with fluorescein (P51) and the Kd was calculated using a quadratic binding model (n = 2 biologically independent experiments). (o) MST analysis of E2-E3 interactions. NEDD4 is labeled with Cy5 at its N-terminus. E2(UbcH5b)-Ub (P49) is generated using the ubiquitin hydrazide mimic method (n = 2 biologically independent experiments).

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Extended Data Fig. 6 MST analysis of HUWE1, E6AP and NEDD4 binding to E2 and NMR strategy to study HECT conformational switch.

(a) MST analysis of UbcH5b E2 binding with HUWE1 HECT or HUWE1 HECT-Ub (P52 and P53). HUWE1 HECT-Ub (P53) was prepared with a ubiquitin Prg probe (n = 3 biologically independent experiments). (b) MST analysis of UbcH5b binding with E6AP or E6AP-Ub (P54 and P55). E6AP-Ub (P55) was prepared with a ubiquitin Prg probe (n = 2 biologically independent experiments). (c) MST analysis of UbcH7 binding with E6AP or E6AP-Ub (P54 and P55) (n = 2 biologically independent experiments). (d) MST analysis of UbcH5b E2 binding to NEDD4 (P36) under high salt (1.6M NaCl) condition. The Kd values were calculated from two repeats using a quadratic binding model (n = 3 biologically independent experiments). (e) MST analysis of UbcH5b E2 binding to NEDD4-Ub (P37) under high salt (1.6M NaCl) condition. The Kd values were calculated from two repeats using a quadratic binding model (n = 3 biologically independent experiments). (f) Ubiquitination assays comparing WT NEDD4 with ΔC-tail-5aa and V588A NEDD4 mutants (P56 and P59). The unmodified NEDD4 band was quantified by densitometry. The percentage of unmodified NEDD4 is listed below each lane (n = 3 biologically independent experiments showed similar results). (g) Semisynthetic scheme for preparing NEDD4 or NEDD4-Ub containing fluoro-Phe896-NEDD4 proteins used in NMR study. (h) Ubiquitination assays of NEDD4 prepared by expressed protein ligation bearing different fluoro-Phe896 regioisomers containing C terminal tails. The NEDD4 proteins containing 2-fluoro-Phe896 (P63), 3-fluoro-Phe896 (P64), and 4-fluoro-Phe896 (P65) were prepared and compared with unligated NEDD4 (aa188-893, P66) for autoubiquitination activity (n = 2 biologically independent experiments showed similar results). (i) Quantification of fluoro-Phe896 containing NEDD4 activity. The ubiquitin western blot signals of the autoubiquitination smear were used to compare the relative activities of these NEDD4 proteins (n = 2 biologically independent experiments showed similar results). (j) A proposed structural model shows the HECT domain undergoes a transition from the T-conformation to the L conformation upon ubiquitin transfer from E2∼Ub to the E3 catalytic Cys. During this T-to-L conformation change, E2 protein is ejected to allow the turnover of the ubiquitination process.

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Extended Data Fig. 7 Maximum likelihood tree from HECT proteins.

Bootstrap values are displayed as black circles. The tree representing the NEDD4 family is labeled and highlighted in red. Human HECT E3 proteins are coloured in red branches.

Extended Data Fig. 8 Unclasped view of a eukaryotic phylogenetic tree shown in Figure 6a.

The leaves of tree represent the species that contain HECT E3 ligases that were used in this study. The nine human NEDD4 HECT E3 ligases are highlighted in red.

Extended Data Fig. 9 NEDD4 family phylogeny tree from maximum likelihood tree analysis.

The species that contain NEDD4 family ligases are listed in this relationship comparison. These species are highlighted in the evolutionary tree of eukaryotes in Figure 6a.

Extended Data Table 1 Chart summary of the affinities (Kd) of various forms of E3 and E3-Ub interacting with E2, substrate or UbV measured with MST or fluorescence anisotropy
Full size table

Supplementary information

Supplementary Information

Supplementary Tables 1–4 and Fig. 1.

Reporting Summary

Supplementary Code 1

NEDD4 HUWE1 hybrid modelling code.

Supplementary Code 2

RSP5-Ub-WBP2 modelling code.

Supplementary Code 3

HECT Most Likelyhood phynogenetic analysis.

Supplementary Data 1

Primers list.

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Jiang, H., Miller, B.D., Viennet, T. et al. Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01576-z

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