Part:BBa_K4165004

Truncated Serine Protease HtrA1

This basic part encodes for Truncated human high temperature requirement A1 which is a serine protease which can degrade a variety of targets including extracellular matrix proteins.

Usage and Biology

The N-terminal domain of homotrimeric HtrA1 has homology to the IGFBP and Kazal proteins, setting it apart from the bacterial HtrA proteases with which it shares a similarity in terms of its trypsin-like catalytic and PDZ domains. The human HtrA1 protein has several different domains, including an N-terminal IGFBP-like module and a Kazal-like module, a protease domain with a trypsin-like structure, and a C-terminal PDZ domain^[1].

The protease domain obtains an allosteric activation signal upon peptide binding via the L3 sensor loop (nomenclature according to refs^[2,3]. The catalytic triad, the oxyanion hole, and the substrate-specificity pockets ^[3,4] all is present in the active conformation of the enzyme, which is achieved by a rearrangement of L3, which in turn triggers a remodeling of the activation domain (loops L1, L2, and LD). The transition in activity is reversible, allowing for fine-tuned and quick regulation in response to specific stress signals, unlike that seen for traditional serine proteases^[4].

Due to the structure and function of HTRA1, we benefited from this system and its features to make our plug sink system.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 72
  
• 1000
  COMPATIBLE WITH RFC[1000]

Features and codon optimized

the length of HTRA1 protein is 480 amino acids which contains: IGFBP N-terminal: 33-100 mediates interaction with TSC2 substrate. Kazal domain: 98-157, Kazal domains are tight binding inhibitors of serine proteases with three conserved disulfide bonds and are exemplified by the pancreatic secretory trypsin inhibitor SPINK1. Serine protease: 204 – 364 PDZ domain: 365- 467, our proteins tau and Amyloid beta will bind to it and activate the catalytic domain when binding peptide binds to it.

                            Figure 1.: this figure shows HTRA1 system and its domains from uniprot.

Source

Q92743 in Uniport. Gene ID: 5654 in NCBI.

Dry Lab

Modeling

After long time of searching , we couldn't find any model for the HTRA1 monomer which contain the whole domains so we decided to model the HTRA1 monomer through multiple modeling tools (Alphafold – Trosetta – Rosettafold – itasser) to get the best model with which we continued the whole project, based on that there was no trimer structure for the HTRA1 on registry and the model which we found as a trimer on RCSB had a problem in the amino acid number specially PDZ domain so we decided to trimerize the HTRA1 on cluspro website to get the final model which we continue the project with it.

              Figure 2.: the Trimer model of HTRA1 as each color is an indicator for each chain visualized by pymol.

Docking

To make sure of each part of the HTRA1 and the whole system, we needed to go through docking, we made docking of the HTRA1 model with the inhibitors to rank the best models of the inhibitors to use (P0C7L1 (BBa_K4165010) and Q8IUB5 (BBa_K4165008)) and to make sure that the inhibitor binds to the Kazal domain.

Then after that we docked the HTRA1 with tau, Amyloid beta, and their binding peptides (BBa_K4165023-BBa_K4165026-BBa_K4165029-BBa_K4165032-BBa_K4165036-BBa_K4165037-BBa_K4165038-BBa_K4165039-BBa_K4165043-BBa_K4165044-BBa_K4165045-BBa_K4165046-BBa_K4165050-BBa_K4165051-BBa_K4165052-BBa_K4165053-BBa_K4165057-BBa_K4165058-BBa_K4165059-BBa_K4165060 for Amyloid-beta and other switches part for tau) to study the structure and the binding affinity with PDZ domain with different linker lengths.

Finally, we docked the Htra1 model with our whole system (from BBa_K4165021 to BBa_K4165050).

References

1- Eigenbrot, C., Ultsch, M., Lipari, M. T., Moran, P., Lin, S. J., Ganesan, R., ... & Kirchhofer, D. (2012). Structural and functional analysis of HtrA1 and its subdomains. Structure, 20(6), 1040-1050.

2- Clausen, T., Southan, C. & Ehrmann, M. Mol. Cell 10, 443–455 (2002)

3- Perona, J.J. & Craik, C.S. J. Biol. Chem. 272, 29987–29990 (1997).

4- Truebestein, L., Tennstaedt, A., Mönig, T., Krojer, T., Canellas, F., Kaiser, M., ... & Ehrmann, M. (2011). Substrate-induced remodeling of the active site regulates human HTRA1 activity. Nature structural & molecular biology, 18(3), 386-388.