Part:BBa_K4165031
HtrA1 switch 11
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), and T7 terminator (BBa_K731721).
Usage and Biology
Switch 11 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 TD28REV and WWW peptides 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 is the inhibitor, which is mainly a serine protease inhibitor, and since our protease is a serine protease, 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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 528
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Dry-lab Characterization
Figure 1. A figure which dsecribes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.
Dry lab
Modeling
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 | 3.76 | 1.5 | 95.17 | 0.69 | -0.31007 | -1.21262 |
Figure 1. The 3D structure of switch 11 visualized by Pymol.
| None |