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Truncated tripartite motif-containing 21 (TRIM21)

An E3 ligase from the TRIM family, which participates in the ubiquitin-proteasome degradation cascade of misfolded proteins.

Usage and Biology

Tripartite motif-containing 21 (TRIM21) is an E3 ubiquitin ligase that has a strong affinity for the Fc domain of antibodies. It is mainly composed of four domains (RING domain, B-box domain, coiled-coil domain, and PRYSPRY antibody-binding region). TRIM21 engages the ubiquitin-proteasome system to destroy antibody-bound pathogens during infection. In our project, we used the truncated version of Trim21 proposed by team NUDT 2020 (BBa_K3396007), they replaced the ‘PRYSPRY’ region with a protein pair (Cohesin 2 and Dockerin S), Cohesin will be fused to Trim21 and Dockerin to our tau binding peptide, resulting in targeting of tau upon binding of the protein pair, followed by its ubiquitination and degradation.

For the degradation to occur, a highly conserved, 76 amino acid polypeptide called ubiquitin must first be activated by an E1 enzyme in an ATP-dependent manner. The E1 binds ubiquitin's C-terminal end to a cysteine residue in its active site through a covalent connection. The E2 or ubiquitin-conjugating enzyme is the second enzyme in the cascade to receive the thioesterified ubiquitin from the E1 active site. Finally, the E3 ubiquitin ligase promotes the transfer of ubiquitin onto the substrate by binding to both the protein substrate and the E2-bound ubiquitin.

                            Figure 1.: A graphical illustration showing the domains of TRIM21. 

Features

Ring-Domain:

B-Box Domain: This domain exerts a regulatory role through interaction with ring domains as it drives the self-assembly of oligomers, bringing together multiple RING domains, which may facilitate RING dimerization.

Coiled-coil Domain: This domain undergoes monomer–dimer exchange to allow homodimerization of TRIM.

Dry Lab Characterization

Optimization

Firstly, we removed the last nucleotide in the original sequence from NUDT team 2020 to avoid the frameshifting, then the sequence was optimized to be expressed in E-coli BL21.

Modeling

Through our work, we modeled Trim-21 once fused to Dockerin and once again to Cohesin, to find out which one of them is more stable when it binds to Trim 21, the structures were modeled by 5 tools (Robetta-iTASSER-Alphafold-Modeller-TR Rosetta) to reach the best model.

The results showed that N-terminal attachment of DocS to Trim-21 is more preferable than that of Coh2, just like it was mentioned in literature. Our top 2 models ranked 5 out of 6 according to our QA code.

                            Figure 2.: Predicted 3D structure of truncated Trim21 model1.

                            Figure 3.: Predicted 3D structure of truncated Trim21 model2.

References

1. Clift, D., McEwan, W. A., Labzin, L. I., Konieczny, V., Mogessie, B., James, L. C., & Schuh, M. (2017). A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell, 171(7), 1692-1706.e18. https://doi.org/10.1016/j.cell.2017.10.033

2. D.L. Mallery, W.A. McEwan, S.R. Bidgood, G.J. Towers, C.M. Johnson, L.C. James Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21) Proc. Natl. Acad. Sci. USA, 107 (2010), pp. 19985-19990

3. Kleiger, G., & Mayor, T. (2014). Perilous journey: a tour of the ubiquitin-proteasome system. Trends in cell biology, 24(6), 352. https://doi.org/10.1016/j.tcb.2013.12.003

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

5. Zhang, Y., Li, L., Munir, M., & Qiu, H. (2018). RING-Domain E3 Ligase-Mediated Host–Virus Interactions: Orchestrating Immune Responses by the Host and Antagonizing Immune Defense by Viruses. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2018.01083

6. Zeng, J., Santos, A. F., Mukadam, A. S., Osswald, M., Jacques, D. A., Dickson, C. F., ... & James, L. C. (2021). Target-induced clustering activates Trim-Away of pathogens and proteins. Nature structural & molecular biology, 28(3), 278-289.