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

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.

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.

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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

Authors

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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The authors declare no competing interests.

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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.

Reporting Summary

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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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