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Target-induced clustering activates Trim-Away of pathogens and proteins

Nature Structural & Molecular Biology volume 28, pages 278–289 (2021)Cite this article

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Abstract

Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyglutamine tracts and expand the Trim-Away toolbox with highly active TRIM21–nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.

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Fig. 1: RING dimerization is required for TRIM21 activation.
Fig. 2: Trim-Away requires recruitment of multiple TRIM21 molecules to the target.
Fig. 3: The nature of the target dictates TRIM21 activation.
Fig. 4: Selective degradation of disease-causing HTT proteins by TRIM21.
Fig. 5: Molecular clustering activates TRIM21.
Fig. 6: New ways to Trim-Away proteins.
Fig. 7: Model for the target-induced clustering mechanism and its applications.

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Data availability

SAXS data are available upon request. All other data generated or analyzed during this study are included in this published article and associated files.

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Schapira, M., Calabrese, M. F., Bullock, A. N. & Crews, C. M. Targeted protein degradation: expanding the toolbox. Nat. Rev. Drug Discov. 18, 949–963 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Yamada, T., Yang, Y. & Bonni, A. Spatial organization of ubiquitin ligase pathways orchestrates neuronal connectivity. Trends Neurosci. 36, 218–226 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Verma, R., Mohl, D. & Deshaies, R. J. Harnessing the power of proteolysis for targeted protein inactivation. Mol. Cell 77, 446–460 (2020).

    Article  CAS  PubMed  Google Scholar 

  5. Wu, T. et al. Targeted protein degradation as a powerful research tool in basic biology and drug target discovery. Nat. Struct. Mol. Biol. 27, 605–614 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. James, L. C., Keeble, A. H., Khan, Z., Rhodes, D. A. & Trowsdale, J. Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function. Proc. Natl Acad. Sci. USA 104, 6200–6205 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mallery, D. L. et al. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21). Proc. Natl Acad. Sci. USA 107, 19985–19990 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Vaysburd, M. et al. Intracellular antibody receptor TRIM21 prevents fatal viral infection. Proc. Natl Acad. Sci. USA 110, 12397–12401 (2013).

    Article  PubMed Central  Google Scholar 

  9. Fan, W. et al. Swine TRIM21 restricts FMDV infection via an intracellular neutralization mechanism. Antiviral Res. 127, 32–40 (2016).

    Article  CAS  PubMed  Google Scholar 

  10. Caddy, S. L. et al. Intracellular neutralisation of rotavirus by VP6-specific IgG. PLoS Pathog. 16, e1008732 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Caddy, S. L. et al. Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity. EMBO J. https://doi.org/10.15252/embj.2020106228 (2020).

  12. Rakebrandt, N., Lentes, S., Neumann, H., James, L. C. & Neumann-Staubitz, P. Antibody- and TRIM21-dependent intracellular restriction of Salmonella enterica. Pathog. Dis. 72, 131–137 (2014).

    CAS  PubMed  Google Scholar 

  13. McEwan, W. A. et al. Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21. Nat. Immunol. 14, 327–336 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. McEwan, W. A. et al. Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation. Proc. Natl Acad. Sci. USA 114, 574–579 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Foss, S. et al. TRIM21—from intracellular immunity to therapy. Front. Immunol. 10, 2049 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. McEwan, W. A., Mallery, D. L., Rhodes, D. A., Trowsdale, J. & James, L. C. Intracellular antibody-mediated immunity and the role of TRIM21. Bioessays 33, 803–809 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Clift, D. et al. A method for the acute and rapid degradation of endogenous proteins. Cell 171, 1692–1706 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Clift, D., So, C., McEwan, W. A., James, L. C. & Schuh, M. Acute and rapid degradation of endogenous proteins by Trim-Away. Nat. Protoc. 13, 2149–2175 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Fletcher, A. J., Mallery, D. L., Watkinson, R. E., Dickson, C. F. & James, L. C. Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21. Proc. Natl Acad. Sci. USA 112, 10014–10019 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kiss, L. et al. A tri-ionic anchor mechanism drives Ube2N-specific recruitment and K63-chain ubiquitination in TRIM ligases. Nat. Commun. 10, 4502 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. de Bie, P. & Ciechanover, A. Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms. Cell Death Differ. 18, 1393–1402 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Peth, A., Uchiki, T. & Goldberg, A. L. ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation. Mol. Cell 40, 671–681 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hofmann, R. M. & Pickart, C. M. In vitro assembly and recognition of Lys-63 polyubiquitin chains. J. Biol. Chem. 276, 27936–27943 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Saeki, Y. et al. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J. 28, 359–371 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kim, H. T. et al. Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages. J. Biol. Chem. 282, 17375–17386 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Ohtake, F., Tsuchiya, H., Saeki, Y. & Tanaka, K. K63 ubiquitylation triggers proteasomal degradation by seeding branched ubiquitin chains. Proc. Natl Acad. Sci. USA 115, E1401–E1408 (2018).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Fletcher, A. J. et al. TRIM5α requires Ube2W to anchor Lys63-linked ubiquitin chains and restrict reverse transcription. EMBO J. 34, 2078–2095 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fletcher, A. J. et al. Trivalent RING assembly on retroviral capsids activates TRIM5 ubiquitination and innate immune signaling. Cell Host Microbe 24, 761–775 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Fiorentini, F., Esposito, D. & Rittinger, K. Does it take two to tango? RING domain self-association and activity in TRIM E3 ubiquitin ligases. Biochem. Soc. Trans. 48, 2615–2624 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Koliopoulos, M. G., Esposito, D., Christodoulou, E., Taylor, I. A. & Rittinger, K. Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity. EMBO J. 35, 1204–1218 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Haubrich, K. et al. RNA binding regulates TRIM25-mediated RIG-I ubiquitylation. Preprint at bioRxiv https://doi.org/10.1101/2020.05.04.070177 (2020).

  32. Sanchez, J. G. et al. TRIM25 binds RNA to modulate cellular anti-viral defense. J. Mol. Biol. 430, 5280–5293 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Diaz-Griffero, F. et al. Rapid turnover and polyubiquitylation of the retroviral restriction factor TRIM5. Virology 349, 300–315 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Li, X. & Sodroski, J. The TRIM5α B-box 2 domain promotes cooperative binding to the retroviral capsid by mediating higher-order self-association. J. Virol. 82, 11495–11502 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yudina, Z. et al. RING dimerization links higher-order assembly of TRIM5α to synthesis of K63-linked polyubiquitin. Cell Rep. 12, 788–797 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ganser-Pornillos, B. K. et al. Hexagonal assembly of a restricting TRIM5α protein. Proc. Natl Acad. Sci. USA 108, 534–539 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Li, Y.-L. et al. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids. Elife 5, e16269 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Dickson, C. et al. Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21. Elife 7, e32660 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  39. McEwan, W. A. et al. Regulation of virus neutralization and the persistent fraction by TRIM21. J. Virol. 86, 8482–8491 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hilpert, K. et al. Anti-c-myc antibody 9E10: epitope key positions and variability characterized using peptide spot synthesis on cellulose. Protein Eng. Des. Sel. 14, 803–806 (2001).

    Article  CAS  Google Scholar 

  41. Krauss, N. et al. The structure of the anti-c-myc antibody 9E10 Fab fragment/epitope peptide complex reveals a novel binding mode dominated by the heavy chain hypervariable loops. Proteins 73, 552–565 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Young, G. et al. Quantitative mass imaging of single biological macromolecules. Science 360, 423–427 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Cole, D., Young, G., Weigel, A., Sebesta, A. & Kukura, P. Label-free single-molecule imaging with numerical-aperture-shaped interferometric scattering microscopy. ACS Photonics 4, 211–216 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kubala, M. H., Kovtun, O., Alexandrov, K. & Collins, B. M. Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci. 19, 2389–2401 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gillingham, A. K. & Munro, S. The PACT domain, a conserved centrosomal targeting motif in the coiled‐coil proteins AKAP450 and pericentrin. EMBO Rep. 1, 524–529 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Shvets, E., Bitsikas, V., Howard, G., Hansen, C. G. & Nichols, B. J. Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids. Nat. Commun. 6, 6867 (2015).

    Article  CAS  PubMed  Google Scholar 

  47. Hayer, A., Stoeber, M., Bissig, C. & Helenius, A. Biogenesis of caveolae: stepwise assembly of large caveolin and cavin complexes. Traffic 11, 361–382 (2010).

    Article  CAS  PubMed  Google Scholar 

  48. Bates, G. P. et al. Huntington disease. Nat. Rev. Dis. Primers 1, 15005 (2015).

    Article  PubMed  Google Scholar 

  49. Owens, G. E., New, D. M., West, A. P. & Bjorkman, P. J. Anti-polyQ antibodies recognize a short polyQ stretch in both normal and mutant huntingtin exon 1. J. Mol. Biol. 427, 2507–2519 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. & Schaffer, D. V. Optogenetic protein clustering and signaling activation in mammalian cells. Nat. Methods 10, 249–252 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Taslimi, A. et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925 (2014).

    Article  CAS  PubMed  Google Scholar 

  52. Park, H. et al. Optogenetic protein clustering through fluorescent protein tagging and extension of CRY2. Nat. Commun. 8, 30 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Kono, K. et al. Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization. Elife 8, e45559 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  54. La Spada, A. R. & Taylor, J. P. Repeat expansion disease: progress and puzzles in disease pathogenesis. Nat. Rev. Genet. 11, 247–258 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Soto, C. & Pritzkow, S. Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. Nat. Neurosci. 21, 1332–1340 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ganser-Pornillos, B. K. & Pornillos, O. Restriction of HIV-1 and other retroviruses by TRIM5. Nat. Rev. Microbiol. 17, 546–556 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Mund, T. & Pelham, H. R. Substrate clustering potently regulates the activity of WW-HECT domain-containing ubiquitin ligases. J. Biol. Chem. 293, 5200–5209 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Słabicki, M. et al. Small-molecule-induced polymerization triggers degradation of BCL6. Nature 588, 164–168 (2020).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  59. Zeng, J., Slodkowicz, G. & James, L. C. Rare missense variants in the human cytosolic antibody receptor preserve antiviral function. Elife 8, e48339 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Liman, E. R., Tytgat, J. & Hess, P. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9, 861–871 (1992).

    Article  CAS  PubMed  Google Scholar 

  61. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  62. Schuck, P. On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal. Biochem. 320, 104–124 (2003).

    Article  CAS  PubMed  Google Scholar 

  63. Brautigam, C. A. in Methods in Enzymology Vol. 562 (ed. Cole, J. L.) 109–133 (Academic Press, 2015).

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Acknowledgements

We thank J. T. Andersson (University of Oslo) for the 9C12 antibody variants and members of the James laboratory for helpful discussions. J.Z. was supported by a PhD Studentship from the Rosetrees Trust and the Frank Edward Elmore Fund (University of Cambridge). M.O. is part of the GABBA PhD program from the University of Porto. D.A.J. was supported by an NHMRC Early Career Fellowship (CJ Martin) (GNT1036521). C.F.D. was supported by an NHMRC Early Career Fellowship (GNT1110116). L.K. is supported by a PhD fellowship from the Boehringer Ingelheim Fonds. W.A.M. is a Lister Institute Prize Fellow and is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant number 206248/Z/17/Z) and is supported by the UK Dementia Research Institute, which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. E.M.-d.-S. acknowledges FCT (Fundação para a Ciência e a Tecnologia) (contract CEECIND/00622/2017 and grant PTDC/BEX-BCM/0432/2014) and project Norte-01-0145-FEDER-000029, supported by NORTE 2020. This work was supported by the MRC (UK, U105181010) and a Wellcome Trust Investigator Award (200594/Z/16/Z).

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Authors and Affiliations

  1. Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK

    Jingwei Zeng, Stephen H. McLaughlin, Christopher M. Johnson, Leo Kiss, Jakub Luptak, Nadine Renner, Marina Vaysburd, Dean Clift & Leo C. James

  2. i3S—Instituto de Investigação e Inovação em Saúde and IBMC Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal

    Ana Filipa Santos, Mariana Osswald & Eurico Morais-de-Sá

  3. Department of Clinical Neurosciences, UK Dementia Research Institute, University of Cambridge, Cambridge, UK

    Aamir S. Mukadam & William A. McEwan

  4. EMBL Australia Node, Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia

    David A. Jacques & Claire F. Dickson

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  1. Jingwei Zeng
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Contributions

D.C. and L.C.J. conceptualized the study; J.Z., M.O., W.A.M., E.M.-d.-S., D.C. and L.C.J. developed the methodology; J.Z., A.F.S., A.S.M., M.O., D.A.J., C.F.D., S.H.M., C.M.J., L.K., J.L., N.R., M.V., W.A.M. and D.C. performed the experiments; D.C. and L.C.J. prepared the manuscript; J.Z., A.F.S., M.O., W.A.M., E.M.-d.-S., D.C. and L.C.J. revised and edited the manuscript; W.A.M., E.M.-d.-S., D.C. and L.C.J. supervised the work; W.A.M., E.M.-d.-S. and L.C.J. acquired funding.

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Correspondence to William A. McEwan, Eurico Morais-de-Sá, Dean Clift or Leo C. James.

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Peer review information Nature Structural & Molecular Biology thanks Alessio Ciulli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 TRIM21 autoubiquitination upon substrate engagement.

a, His-Ubiquitin pulldown from cells expressing the indicated TRIM21 mutants infected with AdV5 ± 9C12 or 9C12(H433A). b-d, TRIM21-His is expressed at endogenous TRIM21 levels (b) and is functional for AdV5 neutralisation (c) and NFκB signalling (d). Graphs in c and d show mean and s.e.m. of n = 3 technical replicates. e, TRIM21-His pulldown from cells infected with AdV5 ± 9C12 or 9C12(H433A). The AdV5 + 9C12 condition was split and one half treated with the deubiquitinase USP2. f, co-crystal structure of TRIM21 RING:Ube2N~ubiquitin complex (PDB: 6S53) showing the tri-anionic anchor motif (E12, E13 and D21) and second site residues (R67 and N71). g, His-Ubiquitin pulldown from cells expressing the indicated TRIM21 mutants infected with AdV5 ± 9C12 or 9C12(H433A). h, His-Ubiquitin pulldown from cells expressing the indicated His-Ubiquitin mutants infected with AdV5 ± 9C12 or 9C12(H433A). *T21 in immunoblots shown on top of panels a, e, g and h shows non-specific binding of unmodified TRIM21 to the NiNTA beads. To ensure equal His-Ubiquitin pulldown between conditions within a single experiment, equal amounts of His-Ubiquitin plasmids were transfected side-by-side for each condition, and His-pulldowns were performed side-by-side under identical conditions (see also Methods). Uncropped blots/membranes are in Supplementary Data 2. Source data for graphs are in Supplementary Data 1.

Extended Data Fig. 2 Further characterisation of TRIM21 RING domain mutants.

a,b, RING dimerisation mutants M10E and M72E are expressed at WT levels (a) and do not affect AdV5 infection efficiency (b). c, His-Ubiquitin pulldown from cells expressing the indicated TRIM21 mutants infected with AdV5 ± 9C12 or 9C12(H433A). *T21 shows non-specific binding of unmodified TRIM21 to the NiNTA beads. e,f, Second site mutants R67A and N71D are expressed at WT levels (e) and do not affect AdV5 infection efficiency (f). g, Neutralisation of AdV5 infection by increasing 9C12 concentrations in cells expressing the indicated TRIM21 mutants. h, AdV5-9C12-induced NFkB activation in cells expressing the indicated TRIM21 mutants. i, Trim-away of target protein IKKα following electroporation (+) of anti-IKKα IgG (anti-IKKα). Immunoblots show IKKα degradation is deficient in cells expressing the N71D mutant TRIM21. Graphs in b,f,g,h show mean and s.e.m for n = 3 independent experiments. Statistical significance is based on one-way ANOVA (b,f,h) and two-way ANOVA (g) and represented with symbols: ns(P>0.05), *(P≤0.05), **(P≤0.01), ***(P≤0.001), ****(P≤0.0001). Uncropped blots/membranes are in Supplementary Data 2. Source data for graphs and statistics are in Supplementary Data 1.

Extended Data Fig. 3 Structural and biophysical characterisation of TRIM21.

a, Schematic of TRIM21 protein sequence indicating RING, B Box, coiled-coil and PRYSPRY domain boundaries. b, SEC-MALS chromatograms of T21CC235 loaded at concentrations of 15 (black), 5 (green), 0.55 (blue) and 0.25 mg/ml (red) in which the refractive index is indicated by the solid lines while the molar mass evaluated from the light scattering analysis is indicated with the corresponding coloured dotted lines. c, Isothermal titration calorimetry (ITC) trace of T21CC235 fitted to a dimer dissociation model reveal an enthalpy of 65 kcal/mol and Kd of 7 µM. d, Analytical ultracentrifugation (AUC) sedimentation velocity analysis of T21RBCC in solution. The upper panel shows absorbance profiles with best fits of a c(s) model (coloured lines) and their residuals to the fits underneath. The different colours represent scans at different times: blue is the earliest time points where very little material has sedimented; through to red where all the material has sedimented and the signal is near baseline across the radius. The lower panel shows the c(s) distribution of specie. T21RBCC sedimented with coefficient of 2.5–2.6S (Sw,20 = 3.3–3.6S) with a frictional ratio of 1.49–1.52 corresponding to a mass of between 52–55 kDa, close to that expected for a dimeric species. e, Concentration-normalised scattering plots and DAM fits from SAXS data for T21CC235 (cyan, χ = 1.00), MBP-T21CC235 (green, χ = 1.31) and T21RBCC (purple, χ = 1.05). f, The linear Guinier regions from (e). f, Derived P(r) curves. The CC235 P(r) curve is consistent with an elongated rod, while the two peaks observed for MBP-T21CC235 and T21RBCC are consistent with dumbbell-shaped molecules. h, Scattering plot with DAM fit from TRIM21:Fc SAXS data (red line, 1.00), TRIM21:Fc atomic model fit (blue, χ = 1.04), and apo-TRIM21 atomic model fit (brown dashed,, χ = 2.00). i, The linear Guinier regions from (h). j, Derived P(r) curves.

Extended Data Fig. 4 Anti-GFP antibodies bind to GFP inside living cells.

RPE-1 TRIM21 KO cells expressing membrane-localised GFP (mem-mEGFP) were electroporated with PBS, control IgG or the indicated anti-GFP antibodies. Cells were fixed 3 hours post-electroporation and stained with alexa 647-conjugated anti-IgG secondary antibodies and imaged by confocal microscopy. Scale bar 10 µm. Representative examples from n = 3 independent experiments.

Extended Data Fig. 5 Proteasomal degradation of GFP-tagged proteins by TRIM21 RING-nanobody fusion.

a-d, NIH3T3-Caveolin-1-GFP (a,b) and RPE-1-H2B-mEGFP-FKBP (c,d) cells were electroporated with mRNA encoding mCherry-T21R-vhhGFP4, incubated with either DMSO (control) or MG132 and imaged (a,c) and GFP fluorescence quantified (b,d) with the IncuCyte system. Time, hours (h) post-electroporation. Scale bars, 20 µm. Error bars show s.e.m. from n = 4 technical replicates. Source data for graphs are in Supplementary Data 1.

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2.

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Supplementary Data 1

Data for graphs and statistics.

Supplementary Data 2

Uncropped blots, gels and membranes.

Supplementary Video 1

Time-lapse video of Drosophila S2 cells expressing the indicated constructs showing that light-induced clustering of mRFP–CRY2clust–TRIM21 triggers protein degradation, as fluorescence loss is inhibited by treatment with a proteasomal inhibitor (MG132). Clustering was triggered by blue light at time 0. Scale bar, 5 μm.

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Zeng, J., Santos, A.F., Mukadam, A.S. et al. Target-induced clustering activates Trim-Away of pathogens and proteins. Nat Struct Mol Biol 28, 278–289 (2021). https://doi.org/10.1038/s41594-021-00560-2

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