Abstract
The E3 ligase TRIM7 has emerged as a critical player in viral infection and pathogenesis. However, the mechanism governing the TRIM7–substrate association remains to be defined. Here we report the crystal structures of TRIM7 in complex with 2C peptides of human enterovirus. Structure-guided studies reveal the C-terminal glutamine residue of 2C as the primary determinant for TRIM7 binding. Leveraged by this finding, we identify norovirus and SARS-CoV-2 proteins, and physiological proteins, as new TRIM7 substrates. Crystal structures of TRIM7 in complex with multiple peptides derived from SARS-CoV-2 proteins display the same glutamine-end recognition mode. Furthermore, TRIM7 could trigger the ubiquitination and degradation of these substrates, possibly representing a new Gln/C-degron pathway. Together, these findings unveil a common recognition mode by TRIM7, providing the foundation for further mechanistic characterization of antiviral and cellular functions of TRIM7.
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Data availability
The structure factors and atomic coordinates for TRIM7 in complex with substrate peptides have been deposited to the Protein Data Bank with accession numbers of 7W0Q (TRIM7–2C, 1.1 Å), 7W0S (TRIM7–2C, 1.4 Å), 7W0T (TRIM7–2C, 1.57 Å), 7X6Y (TRIM7–NSP5, 1.39 Å), 7X70 (TRIM7–NSP8, 1.25 Å) and 7X6Z (TRIM7–NSP12, 1.43 Å). Source data are provided with this paper.
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Acknowledgements
We thank J. Lei for supporting plasmids encoding SARS-CoV-2 proteins. We thank the staffs from BL17B/BL18U1/BL19U1/BL19U2/BL01B beamlines of the National Facility for Protein Science in Shanghai at Shanghai Synchrotron Radiation Facility, for assistance during data collection. This work is supported by the National Natural Science Foundation of China Grants (32071218 to H.Z.) and the National Key Research and Development Program of China (2018YFC1313002 to L.L.).
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X.L., Y.W., Y.M. and Y.Z. performed the sample preparation, biochemistry and crystallization experiments. Xu.L. collected the diffraction data and solved the structure with the help from X.L. and Y. M. X.C. performed the molecular dynamics analysis. J.X., Y. Li., Y. Lu, X.L., Z.L., X.Z., D.J. and P.W. carried out the cellular experiments. C.X., L.L., G.Y. and H.Z. analyzed the data. H.Z., G.Y. and L.L. designed the project and wrote the manuscript.
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X.C. is the founder of YDS Pharmatech. The remaining authors declare no competing interests.
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Nature Chemical Biology thanks Chao Xu, Matthew Ravalin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 TRIM7 interacts with enteroviruses 2C.
a, Sequence alignment of 2C proteins from different enteroviruses. The last eight residues at the C-terminus are displayed. Invariant and conserved residues are shaded green and yellow, respectively. b, ITC measurements of peptides derived from the C-terminal fragments of 2C proteins from different enteroviruses, including enterovirus 71, poliovirus and enterovirus D68, to TRIM7PRY-SPRY.
Extended Data Fig. 2 Structural features of the TRIM7PRY-SPRY-2C peptide complex.
a, The overall structures of TRIM7PRY-SPRY-2C peptide complex solved in different space groups (top). Bottom, 2Fo-Fc electron density map of the 2C fragments contoured at 1.0 σ. b, Structural comparison of the three TRIM7PRY-SPRY-2C peptide structures and a previously published substrate-free structure (PDB ID: 6UMA). c, ITC measurement of TRIM7PRY-SPRY-2CΔN association in a high salt buffer containing 500 mM NaCl. d, Conservation scores across different species were mapped to the TRIM7 structure. Multiple sequence alignment of human TRIM7 and orthologs in other mammals, birds and amphibians were performed with Clustal Omega. The multiple sequence alignment and the resolved structure of TRIM7PRY-SPRY were used as inputs for conservation analysis using the ConSurf web server (https://consurf.tau.ac.il/).
Extended Data Fig. 3 Quantification of the binding affinity between TRIM7 and CVB3 2C mutations.
Thermodynamic analysis of the interaction between TRIM7PRY-SPRY and 2C mutants (T323G, T323S, T323I).
Extended Data Fig. 4 MD simulations and ITC measurements of the interaction between TRIM7PRY-SPRY and 2C variants.
a, (left) Structural comparison between TRIM2-2C and the calculated TRIM7-2C (Q329E). (right) Effects of simulations on the Q329E mutation. Molecular dynamics simulation suggested that Q329E mutation eliminates the interactions between the residue 329 and TRIM7, especially the interactions with Arg385, Asn438, and Ser499 in TRIM7 (Supplementary Table 2). Consequently, the C-terminal carboxylate group of 2C at residue 329 is more exposed to solvent, leading to a further reduced TRIM7-2C interaction and increased 2C C-terminal solvation. b, The root-mean-square deviation (RMSD) change of the 2C peptide structures in MD simulation (left). The RMSD values of WT (black) and Q329E (red) 2C peptide structures with reference to the first frame of the trajectory in MD simulation were calculated and plotted. While the WT 2C peptide gradually converged to the original position, the Q329E mutant drifted away as indicated by the increasing RMSD over MD simulation. Right, the Q329E mutation led to increased hydrogen bonding with water. The number of hydrogen-bonded water molecules to the side chain amide of Q329 in WT 2C (black) and the side chain carboxyl of E329 in the mutant (red) were plotted over MD simulation.
Extended Data Fig. 5 NSP proteins from SARS-CoV-2 counteract the IFNB1 promoter activity.
a, Co-immunoprecipitation of TRIM7 with NSP5 and NSP8. FLAG-tagged NSP5, NSP8 or empty vector was co-transfected with HA-TRIM7 in HEK293T cells. Cells were lysed and immunoprecipitated with anti-HA antibody and detected by anti-FLAG antibody. HA-TRIM7-Mut, TRIM7 catalytic dead mutant (C29A/C31A); NSP5/NSP8-Q/A, the alanine replacement of the C-terminal glutamine in NSP5/8. b, Crystal structure of TRIM7PRY-SPRY in complex with NSP12 peptide (aa 928-932). c, 2Fo-Fc electron density map of the TRIM7-bound NSP peptides contoured at 1.0 σ. d, Luciferase activity of IFNB1 promoter reporter in HEK293T cells transfected with indicated NSP proteins and RIG-I CARD expression plasmids. Relative luciferase activity was quantified 24 h post-transfection. Data are presented as mean ± SEM; n = 6, biologically independent samples were examined over three independent experiments. Statistical significance was determined by comparing with ‘Vector’ group using one-way ANOVA with Tukey’s correction. e, f, Analysis of TBK1 phosphorylation in the presence of NSP5 (e) or NSP8 (f).
Extended Data Fig. 6 ITC measurements of TRIM7PRY-SPRY with multiple cellular proteins functionally linked to TRIM7.
a, TRIM7PRY-SPRY binds with GN1 and RACO-1 C-terminus peptide fragments. b, Thermodynamic analysis of the interaction between TRIM7PRY-SPRY and BRMS1 peptide (left panel), DUSP6 peptide (middle panel) and STING protein (right panel).
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Supplementary Figs 1–11, Supplementary Tables 1–3 and uncropped gel images.
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Source Data Fig. 4
Unprocessed western blots.
Source Data Fig. 5
Unprocessed western blots.
Source Data Fig. 6
Unprocessed western blots.
Source Data Extended Data Fig. 5
Unprocessed western blots.
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Liang, X., Xiao, J., Li, X. et al. A C-terminal glutamine recognition mechanism revealed by E3 ligase TRIM7 structures. Nat Chem Biol 18, 1214–1223 (2022). https://doi.org/10.1038/s41589-022-01128-x
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DOI: https://doi.org/10.1038/s41589-022-01128-x
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