Part:BBa_K4165059
HtrA1 Switch 39
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_K4165017), seed peptide (BBa_K4165012), GS Linker (BBa_K4165019), seed peptide (BBa_K4165012), GS Linker (BBa_K4165017), WAP inhibitor (BBa_K4165008), and T7 terminator (BBa_K731721).
Usage and Biology
Switch 39 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 seed peptide which is considered as an amyloid binding peptide 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 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
- 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
Modeling
The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta.
| cbeta_deviations | clashscore | molprobity | ramachandran_favored | ramachandran_outliers | Qmean_4 | Qmean_6 |
|---|---|---|---|---|---|---|
| 0 | 154.75 | 2.65 | 98.44 | 0 | -1.38724 | -2.76799 |
Figure 1. The 3D structure of switch 39 modelled by tRrosseta.
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
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