Part:BBa_K4165202
Trim-(GGGGS)3-Docs
This parts code for the Trim21 E3 ligase with the PRYSPRY domain truncated (BBa_K3396007), fused to type 1 Dockerin module derived from Clostridium thermocellum cellulosome scaffoldin by a Glycine Serine flexible linker repeated three times to maintain part flexibility needed during target ubiquitination.
Usage and Biology
This fusion protein is part of our Trim System (Snitch System), it is a modified version of the NUDT 2020 part (BBa_K3396005). It is supposed to bind to the PROTAC (Coh2-linker-tau_binding_peptide) to assemble a full system that will be able to target Tau protein through the binding peptide and recruit ubiquitin to tag the whole protein to initiate degradation by proteasomes through Trim21.
Trim21 (E3) is an integral part of the protein turnover processes which serves as a quality control step. In order to be degraded by proteasome 26S, the protein must be tagged with a ubiquitin tail. E3 ligase serves the function of transferring the ubiquitin to the protein of interest. So, in order for the process to be directed more specifically at certain proteins, we took advantage of the high affinity between the two modules (DocS and Coh2) which make up the cellulosome, to act as a protein pair that will guide trim to any chosen protein.
This process could be done by anchoring Trim21 to either one of the modules and anchoring its other counterpart with a targeting domain for the protein of interest, more feasible and directed ubiquitination of the target protein has been achieved, which eventually leads to specific degradation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 535
Improvement
There seemed to be some mistakes in the original sequence uploaded by NUDT 2020 (BBa_K3396005), there was an additional stop codon in the middle of the fusion protein and an extra Tyrosine residue in their Glycine-Serine Linker. These misconceptions hinder the usage of this part by any iGEM team, so we ought to provide them with the correct sequence that they could use easily if they want to assemble this part.
Figure 1.: Alignment of modified Trim-G4S-DocS sequence with the original sequence from of NUDT_2020 (BBa_K3396007) showing the extra amino acid residues (Valine-Leucine-Glutamic acid- Lysine) and the stop codon in the middle.
Modeling
tTrim21-(G4S)3-DocS is modeled by AlphaFold2, ITASSER, MODELLER, Robetta and TrRosetta, best model obtained from TrRosetta. We assembled this part with different linker and substituted the DocS with Coh2 (BBa_K4165200), (BBa_K4165201), and (BBa_K4165203). The results of model assessment showed that this part is the best one.
Figure 1.: Predicted 3D structure of our fusion protein tTrim21-(G4S)3-DocS.
Table 1: Quality assessment parameters of tTrim21-(G4S)3-DocS. model.
The modeling results show that fusing DocS to tTrim21 by (G4S)3 linker improves the quality of the model than DocS basic part (BBa_K3396000).
1.2. Docking
Docking is done to test the interaction of the whole fusion proteins together, and how it can change the binding affinity from the tagged part in our contribution to DocS (BBa_K3396000). The results prove that when DocS is fused to tTrim21, the resulted protein has higher affinity to GST-Coh2-linker-WWW than basic DocS and Coh2.
Figure 2.: All docked structure of TLD by Galaxy and ClusPro displayed by Pymol.
Table 2: Binding affinity of tTrim21-(G4S)3-DocS to protacs with different tau binding peptides models.
WetLab Results
Transformation of His Trim21 (L) Doc in BL-21 using pGS-21a
Figure 2. Transformed plate of His Trim21 (L) Doc + pGS-21a
Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET vector
Figure 3. Transformed plate of His Trim21 (L) Doc + pJET
Comparison between chemical lysis and sonication for His Trim21 (L) DOC
Figure 4. This graph shows a significant difference between chemical lysis and sonication
for His Trim21 (L) DOC, after we had the results, we optimized our protocol to
use sonication for His Trim21 (L) DOC
SDS PAGE of induced and non-induced samples of His Trim 21 (L) DOC
Figure 5. This figure shows the comparison between the induced and non-induced samples of His Trim21
(L) DOC, where well no.1 is the non-induced sample while well no.3 is the induced sample showing that
our protein is induced effectively owing to our right choice of IPTG, time interval and concentration
Pull down assay of His Trim21 (L) DOC against GST COH WWW and GST COH TD28 Rev
Figure 6. This graph shows the comparison of pull down assay between His Trim (L) DOC against GST COH WWW and
GST COH TD28 Rev, showing that the interaction between His Trim21 (L) DOC and GST COH WWW is better than that
of His Trim (L) DOC and GST COH TD28 Rev as the concentration of the elution coming from the pull down assay
of His Trim21 (L) DOC and GST COH WWW is more than that of His Trim21 (L) DOC and GST COH TD28rev
References
1- Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH. Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. J Mol Biol. 2001 Mar 30;307(3):745-53. doi: 10.1006/jmbi.2001.4522. PMID: 11273698.
2- Ronchi, V. P., & Haas, A. L. (2012). Measuring rates of ubiquitin chain formation as a functional readout of ligase activity. In Ubiquitin Family Modifiers and the Proteasome (pp. 197-218). Humana Press.
3- Collins, G. A., & Goldberg, A. L. (2017). The logic of the 26S proteasome. Cell, 169(5), 792-806.
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