HtrA1 switch 2

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), SPINK8 Inhibitor (BBa_K4165010), TD28rev (BBa_K4165006), WWW (BBa_K4165007), H1A peptide (BBa_K4165000) and T7 terminator (BBa_K731721).

Usage and Biology

Switch 2 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 PDZ peptide, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 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 TD28REV and WWW peptides which are considered as tau binding peptides are proved experimentally to bind with tau inhibit the aggregations of tau aggregations respectively. The H1A peptide was also proven 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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Dry-lab Characterization

The top model is TrRoseeta model and the workflow to reach the model will be described below.

                      Figure 1.:3D Predicted Structure of Switch 2 Protein by Pymol Visualization.

Figure 2. A figure which describes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.

Switch construction Pipeline

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, please check our Modeling page.

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.

10) Assembly

p style=" font-weight: bold; font-size:14px;">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.

abc

        Figure (a,b,c) : 3D structure of  P0C7L1 Inhibitor , H1A Peptide ,  and TD28rev-GGSGGGG-WWW clamp 
                                    used in our assembly of switch 2

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 <a href="https://2022.igem.wiki/cu-egypt/ProteinModelling.html">Modeling page</a>.

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 <a href="https://2022.igem.wiki/cu-egypt/ProgrammingClub.html">Programming club</a> under the name of Modric.

13) Ranking

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: (Link software page) under the name of Modric.

The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta with a score of 4 out of 6 according to our quality assessment code.

cbeta_deviations	clashscore	molprobity	ramachandran_favored	ramachandran_outliers	Qmean_4	Qmean_6
0	4.2	1.34	97.24	0.69	0.006651	-1.22218

                               Table 1: Quality assessment parameters of Switch 2 model.

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. Goedert, M., & Spillantini, M. G. (2017). Propagation of Tau aggregates. Molecular Brain, 10. https://doi.org/10.1186/s13041-017-0298-7

2. Etienne, M. A., Edwin, N. J., Aucoin, J. P., Russo, P. S., McCarley, R. L., & Hammer, R. P. (2007). Beta-amyloid protein aggregation. Methods in molecular biology (Clifton, N.J.), 386, 203–225. https://doi.org/10.1007/1-59745-430-3_7

4. Seidler, P., Boyer, D., Rodriguez, J., Sawaya, M., Cascio, D., Murray, K., Gonen, T., & Eisenberg, D. (2018). Structure-based inhibitors of tau aggregation. Nature chemistry, 10(2), 170. https://doi.org/10.1038/nchem.2889

5. 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.