Part:BBa_K4165052
HtrA1 Switch 32
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), GS Linker (BBa_J18921), seed peptide (BBa_K4165012), GS Linker (BBa_K4165019), seed peptide (BBa_K4165012), GS Linker (BBa_J18921), WAP inhibitor (BBa_K4165008), and T7 terminator (BBa_K731721).
Usage and Biology
Switch 32 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 589
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 572
Illegal AgeI site found at 308 - 1000COMPATIBLE WITH RFC[1000]
Dry-lab Characterization
Figure 1. A figure which dsecribes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.
Modeling
The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta.
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 modelling (ab initio) and template-based modelling. For modelling 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 modelling 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 of inhibitor and HTRA Binding Peptide are docked with HtrA1 (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 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 binded to the same site or not.
Figure 2. 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 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.
a
b
c
Figure 3. a) Seed_GGSGGGGG_seed clamp b) HTRA Binding Peptide 1 c)WAP-four disulfide core domain 13 serine protease inhibitor.
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 Abu Trika.
Table 1. quality assessment parameters of switch 32.
| cbeta_deviations | clashscore | molprobity | ramachandran_favored | ramachandran_outliers | Qmean_4 | Qmean_6 |
|---|---|---|---|---|---|---|
| 0 | 2.15 | 1.46 | 92.31 | 0.77 | -1.05267 | -1.3236 |
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.
Figure 4. The 3D structure of switch 32 top model modelled by tRrosetta.
Table 2. RMSD calculated from alignment of switch 32 and its basic parts.
| average RMSD from free HtrA binding peptide1 | average RMSD from docked HtrA binding peptide1 | RMSD from free seed peptide |
|---|---|---|
| 2.044 | 1.861 | 7.5 |
| None |