HtrA1 Switch number 12

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), GS Linker (BBa_K4165017), seed peptide (BBa_K4165012), GS Linker (BBa_K4165019), seed peptide (BBa_K4165012), GS Linker (BBa_K4165017), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).

Usage and Biology

Switch 12 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 accoding to both CAPRI and NCBI protocols.

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

Modeling

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

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        Figure (a,b,c) : 3D structure of  P0C7L1  Inhibitor , H1A Peptide ,  and Seed-GGSGGGGG-Seed clamp 
                                    used in our assembly of switch 12

Docking

switch12 vs HtrA1 trimer:

ΔG = -22.315

                                    Figure 2. The 3D structure of switch 12 docked to HtrA1

Mathematical modeling

Transcription rate and translation rate under T7 promotor

the mathematical modeling was based on our code for the calculation of transcription and translation (you can find it in the code section) beside with the estimated results from the wet lab.

                   Figure 3. this figure shows the results from the transcription and translation code showing 
                                the variation of mRNA and protein concentrations with time.

Dry-lab Characterization

                   Figure 4. A figure which dsecribes 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.

9) Linker length

The linker lengths are acquired by seeing the distance between the inhibitor and the HtrA1 binding peptide which is between both C terminals, N terminals, C- and N- terminal, and N- and C-terminals.

10) Assembly

After settling on the linkers lengths, now we will proceed to the assembly step of the whole system which is done using TRrosetta, AlphaFold, RosettaFold, and Modeller.

            Figure 5: 3D Structure of Clamp Seed-GGSGGGGG-Seed used in our assembly of switch 12

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.

Table 1: Quality assessment parameters of Switch 12 model.

cbeta_deviations	clashscore	molprobity	ramachandran_favored	ramachandran_outliers	Qmean_4	Qmean_6
0	2.12	0.98	98.51	0/td>	0.149	-1.63

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.

RMSD Before Docking	RMSD After Docking
1.29	1.66

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.

Refernces

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.2113520119

5. 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.