Difference between revisions of "Part:BBa K4165041"
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<p style=" font-weight: bold; font-size:14px;">11) Structure Assessment</p> | <p style=" font-weight: bold; font-size:14px;">11) Structure Assessment</p> | ||
| − | <p>In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall | + | <p>In order to assess the quality of our structures, we used the Swiss-Model tool, which gives an overall quality of any 3D structure (For more information, please check our (<a href="https://2022.igem.wiki/cu-egypt/ProteinModelling.html">Modeling page </a>).</p> |
<p style=" font-weight: bold; font-size:14px;">12) Quality Assessment </p> | <p style=" font-weight: bold; font-size:14px;">12) Quality Assessment </p> | ||
| − | <p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information | + | <p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information, please proceed to our (<a href="https://2022.igem.wiki/cu-egypt/ProgrammingClub.html">Programming club </a>) under the name of Modric.</p> |
Revision as of 00:21, 14 October 2022
HtrA1 Switch 21
This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), H1A (BBa_K4165000), GGGGS linker (BBa_K4165068), Seed Peptide (BBa_K4165012), GGSGGGG Linker (BBa_K4165018), Seed Peptide (BBa_K4165012), GGGGS Linker (BBa_K4165068), WAP inhibitor (BBa_K4165008), and T7 terminator (BBa_K731721)
Usage and Biology
Switch 21 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 peptide binding PDZ, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 upon clamp binding to the target protein requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.
The seed peptide is considered as an amyloid binding peptide and is proved experimentally to inhibit the aggregations of amyloid beta through cell viability assays with a survival rate values nearly 100%. The H1A peptide was validated to bind with the PDZ of HtrA1 experimentally. The last part, which is the inhibitor, which is mainly a serine protease inhibitor, and since our protease is a serine protease, so it will act and inhibit the Protein. The whole construction was similarly proved from literature .The process of assembly of the whole switch was done according to both CAPRI and NCBI protocols.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 499
Illegal AgeI site found at 235 - 1000COMPATIBLE WITH RFC[1000]
Dry-Lab Characterization
Top model
Figure 1. The 3D structure of Top model of switch 21 modeled by TRrosetta.
The pipeline for creating this model is discussed in detail in the section below:
Switch construction Pipeline
Figure 2. A figure which describes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.
1) Modelling
Since our Switch parts (HTRA1 binding peptide, TAU, and Beta-amyloid Binding peptide) do not have experimentally acquired structures, we modeled each one of them separately. This approach is done using both denovo modeling (ab initio) and template-based modeling. For modeling small peptides of our system, we used AppTest and Alphafold.
2) Structure Assessment
In order to assess the quality of generated structures, we used the Swiss-Model tool, which gives an overall quality of any 3D structure (For more information: Modeling .
3) Quality Assessment
Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models out of score 6. For more information: Programming club page code under the name of Modric..
4) Filtering
We take the top-ranked models from the previous steps that have either a score of 5 or 6
5) Docking
The top models of inhibitor and HTRA Binding Peptide are docked with HtrA1, and the top models of the clamps are docked with the Target protein, that is, in our case is Beta-amyloid (BBa_K4165004).
6) Ranking
The docking results are ranked according to the Delta free energy generated by PRODIGY. For more information please check our Docking page.
7) Top Models
The results from PRODIGY are ranked, and the top three models are chosen after the models are visualized to ensure that the proteins interact at the right designated domain to proceed with the next step. For more information please check our Docking page.
8) Alignment
Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex, the HtrA1-inhibitor complex, to check whether they bonded to the same site.
Figure 3. Aligned structures of HtrA1 binding peptide 1 docked to HtrA1 and inhibitor docked to HtrA1.
9) Linker length
The linker lengths are acquired by seeing the distance between the inhibitor and the HtrA1 binding peptide between both C terminals, N terminals, C- and N- terminal, and N- and N- and C-terminals the linker length is calculated to be between 12.8 and 24.7 angstroms.
10) Assembly
After settling on the linkers' lengths, we will now proceed to the assembly step of the whole system, which is done using TRrosetta, AlphaFold, RosettaFold, and Modeller.
a
b
c
11) Structure Assessment
In order to assess the quality of our structures, we used the Swiss-Model tool, which gives an overall quality of any 3D structure (For more information, please check our (Modeling page ).
12) Quality Assessment
Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information, please proceed to our (Programming club ) under the name of Modric.
| cbeta_deviations | clashscore | molprobity | ramachandran_favored | ramachandran_outliers | Qmean_4 | Qmean_6 |
|---|---|---|---|---|---|---|
| 0 | 6.33 | 1.7 | 94.89 | 0.73 | -1.65604 | -2.4253 |
14) Alignment
The docked structures are then aligned and compared to the basic parts, which are docked with the protein of interest (HtrA1). The structures with the least RMSD are chosen following the recommended range provided by CAPRI protocol.
Conclusion
The top model was HtrA1 switch 12 (BBa_K4165032) since it was the best switch fulfilling the criteria of structure assessment, docking, and RMSD.References
1. Lu, J., Cao, Q., Wang, C., Zheng, J., Luo, F., Xie, J., ... & Li, D. (2019). Structure-based peptide inhibitor design of amyloid-β aggregation. Frontiers in molecular neuroscience, 12, 54. 2. Romero-Molina, S., Ruiz-Blanco, Y. B., Mieres-Perez, J., Harms, M., Münch, J., Ehrmann, M., & Sanchez-Garcia, E. (2022). PPI-Affinity: A Web Tool for the Prediction and Optimization of Protein–Peptide and Protein–Protein Binding Affinity. Journal of Proteome Research. 3. Stein, V., & Alexandrov, K. (2014). Protease-based synthetic sensing and signal amplification. Proceedings of the National Academy of Sciences, 111(45), 15934-15939 4. 7. Rey, J., Breiden, M., Lux, V., Bluemke, A., Steindel, M., & Ripkens, K. et al. (2022). An allosteric HTRA1-calpain 2 complex with a restricted activation profile. Proceedings Of The National Academy Of Sciences, 119(14). doi: 10.1073/pnas.21135201195. Santos, L. H., Ferreira, R. S., & Caffarena, E. R. (2019). Integrating molecular docking and molecular dynamics simulations. In Docking screens for drug discovery (pp. 13-34). Humana, New York, NY.