Revision as of 03:46, 14 October 2022

HtrA1 switch 7

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 7 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.

Sequence and Features

Dry Lab

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

                      Figure 1.:3D Predicted Structure of Switch 7 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 parts do not have experimentally acquired structures, we have to model them. 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 our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (Link 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 For more information: (Link software page) under the name of Modric.

4) Filtering

We take the top-ranked models from the previous steps.

5) Docking

The top models are docked with the protein of interest (in our case it was the HtrA1 with a BBa_K4165004.

6) Ranking

The docking results are ranked according to their PRODIGY results. For more information: (Link Docking page).

7) Top Models

The results that came out from PRODIGY are ranked and the top models are chosen to proceed with to the next step. For more information: (Link Docking page).

8) Alignment

Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex which is the HtrA1-inhibitor complex to check whether they bound to the same site or not.

     Figure 3. Aligned structures of HtrA1 binding peptide 1 docked to HtrA1 and inhibitor docked to HtrA1. </p>

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.

                            Table 1: The average linker amino acid length of all possibilities . 

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.

11) Structure Assessment

In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (Link modelling 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: (Link software page) 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 5 out of 6 according to our quality assessment code.

                              Table 2: Quality assessment parameters of Switch 3 model. 

14) Alignment

The docked structures are then aligned and compared to the basic parts which are docked with protein of interest (HtrA1). The structures with least RMSD are chosen.

Conclusion

The top model was HtrA1 switch 10 (BBa_K4165030) since it was the best switch fulfilling the criteria of structure assessment, docking, and RMSD.