Difference between revisions of "Part:BBa K4165021"
 
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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).
 
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===
 
===Usage and Biology===
−
Switch 11 is used to mediate the activity of HTRA1. 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 affinity clamp for tau and beta-amyloid (both domains of affinity clamp are connected with linker2) binding and stabilizing the inhibitor away from the active site these two domains (inhibitor and affinity clamp connected with linker1). Additionally, (H1A) binding peptide bound to the PDZ domain and connected to the affinity clamp on the other side with linker3.
+
Switch 1 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 TD28REV and WWW peptides which are 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, 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. The process of assembly of the whole switch was done accoding to both CAPRI and NCBI protocols.
 +
 +
 
 
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===Modeling===
+
 
−
TRrosetta models this composite part with a score of 5 out of 6. The ranking code (you can find the python script file on the programming club page with further explanation of how you can optimize it to your needs). This code works by ranking the predicted 3D structures and scoring them out of 6. In addition, we considered the structural assessment guidelines since the evaluation of the predicted structure depends on the values of Ramachandran Plots, MolProbity, Clash Score, C-Beta Deviations, and QMEAN 4,6.
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<h2 style="text-weight: bold;">Sequence and Features</h2>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4165021 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4165021 SequenceAndFeatures</partinfo>
  
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===Dry-lab Characterization===
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<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/registry/dry-lab-modelling-pipeline.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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 +
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                    Figure 1. A figure which dsecribes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.
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 +
<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
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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.
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switch1.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                          Figure.1: This figure shows the Switch 1 top model by Pymol visualization.
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              <p style=" font-weight: bold; font-size:13px;"> Table 1: Quality assessment parameters of Switch 1 model. </p>
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<html>
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<style>
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table, th, td {
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  border:1px solid black; margin-left:auto;margin-right:auto;
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}
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</style>
 +
<body>
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<table style="width:65%">
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<table>
 +
  <tr>
 +
    <th>cbeta_deviations</th>
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    <th>clashscore</th>
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    <th>molprobity</th>
 +
    <th>ramachandran_favored</th>
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    <th>ramachandran_outliers</th>
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    <th>Qmean_4</th>
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    <th>Qmean_6</th>
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  </tr>
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  <tr>
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    <td>0</td>
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    <td>4.23</td>
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    <td>1.2</td>
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    <td>99.3</td>
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    <td>0.7</td>
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    <td>0.384058</td>
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    <td>-0.7745</td>
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  </tr>
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</table>
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</body>
 +
</html>
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 +
<h1>Switch construction Pipeline</h1>
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<html>
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<p style=" font-weight: bold; font-size:14px;"> 1) Modelling </p>
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<p> Since our Switch parts (HTRA1 binding peptide, TAU, and Beta-amyloid Binding peptide) do not have experimentally acquired structures, we modeled each one of them separately. 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.</p>
 +
 +
<p style=" font-weight: bold; font-size:14px;"> 2) Structure Assessment </p>
 +
 +
<p>In order to assess the quality of generated structures, we used the Swiss-Model tool, which gives an overall quality of any 3D structure (For more information, please check our 
 +
<a href="https://2022.igem.wiki/cu-egypt/ProteinModelling.html">Modeling page</a>.</p>
 +
 +
<html>
 +
<p style=" font-weight: bold; font-size:14px;"> 3) Quality Assessment </p>
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<p>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 out of score 6. For more information: <a href="https://2022.igem.wiki/cu-egypt/ProgrammingClub.html">Programming club page code under the name of Modric.</a>.</p>
 +
 +
<p style=" font-weight: bold; font-size:14px;">4) Filtering</p>
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<p>We take the top-ranked models from the previous steps that have either a score of 5 or 6 </p>
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<p style=" font-weight: bold; font-size:14px;">5) Docking</p>
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<p>The top models of inhibitor and HTRA Binding Peptide are docked with HtrA1, and the top models of the clamps are docked with the Target protein, that is, in our case is Beta-amyloid (BBa_K4165004).</p>
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 +
<html>
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<p style=" font-weight: bold; font-size:14px;">6) Ranking</p>
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<p>The docking results are ranked according to the Delta free energy generated by PRODIGY. For more information please check our <a href="https://2022.igem.wiki/cu-egypt/Docking.html">Docking page</a>.</p>
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 +
<html>
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<p style=" font-weight: bold; font-size:14px;">7) Top Models</p>
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<p>The results from PRODIGY are ranked, and the top three models are chosen after the models are visualized to ensure that the proteins interact at the right designated domain to proceed with the next step. For more information please check our <a href="https://2022.igem.wiki/cu-egypt/Docking.html">Docking page</a>.</p>
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<p style=" font-weight: bold; font-size:14px;">8) Alignment</p>
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<p>Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex, the HtrA1-inhibitor complex, to check whether they bonded to the same site.</p>
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 +
 +
<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/switch31/picture10.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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<p style="text-align:center;"> Figure 3. Aligned structures of HtrA1 binding peptide 1 docked to HtrA1 and inhibitor docked to HtrA1. </p>
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 +
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<p style=" font-weight: bold; font-size:14px;">9) Linker length</p>
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<p>The linker lengths are acquired by seeing the distance between the inhibitor and the HtrA1 binding peptide between both C terminals, N terminals, C- and N- terminal, and N- and N- and C-terminals.
 +
 +
 +
 +
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<p style=" font-weight: bold; font-size:14px;">10) Assembly</p>
 +
<p>After settling on the linkers' lengths, we will now proceed to the assembly step of the whole system, which is done using TRrosetta, AlphaFold, RosettaFold, and Modeller.</p>
 +
 +
<html>
 +
<p style=" font-weight: bold; font-size:14px;">11) Structure Assessment</p>
 +
<p>In order to assess the quality of our structures, we used the Swiss-Model tool, which gives an overall quality of any 3D structure (For more information, please check our <a href="https://2022.igem.wiki/cu-egypt/ProteinModelling.html">Modeling page</a>.</p>
 +
 +
<html>
 +
<p style=" font-weight: bold; font-size:14px;">12) Quality Assessment </p>
 +
<p>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, please proceed to our <a href="https://2022.igem.wiki/cu-egypt/ProgrammingClub.html">Programming club</a> under the name of Modric.</p>
 +
 +
 +
<table style="width:65%">
 +
<table>
 +
  <tr>
 +
    <th>cbeta_deviations</th>
 +
    <th>clashscore</th>
 +
    <th>molprobity</th>
 +
    <th>ramachandran_favored</th>
 +
    <th>ramachandran_outliers</th>
 +
    <th>Qmean_4</th>
 +
    <th>Qmean_6</th>
 +
  </tr>
 +
  <tr>
 +
    <td>0</td>
 +
    <td>6.33</td>
 +
    <td>1.7</td>
 +
    <td>94.89</td>
 +
    <td>0.73</td>
 +
    <td>-1.65604</td>
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    <td>-2.4253</td>
 +
  </tr>
 +
</table>
 +
 +
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<p style=" font-weight: bold; font-size:14px;">14) Alignment</p>
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<p>The docked structures are then aligned and compared to the basic parts, which are docked with the protein of interest (HtrA1). The structures with the least RMSD are chosen following the recommended range provided by CAPRI protocol.</p>
 +
 +
<html>
 +
<style>
 +
table, th, td {
 +
  border:1px solid black; margin-left:auto;margin-right:auto;
 +
}
 +
</style>
 +
<body>
 +
<table style="width:65%">
 +
<table>
 +
  <tr>
 +
    <th>RMSD Before Docking</th>
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    <th>RMSD After Docking</th>
 +
 +
  </tr>
 +
  <tr>
 +
    <td>1.41</td>
 +
    <td>1.85</td>
 +
 +
  </tr>
 +
</table>
 +
</body>
 +
</html>
 +
 +
 +
===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.
 +
 +
===References===
 +
1. Goedert, M., & Spillantini, M. G. (2017). Propagation of Tau aggregates. Molecular Brain, 10. https://doi.org/10.1186/s13041-017-0298-7
 +
 +
2. Etienne, M. A., Edwin, N. J., Aucoin, J. P., Russo, P. S., McCarley, R. L., & Hammer, R. P. (2007). Beta-amyloid protein aggregation. Methods in molecular biology (Clifton, N.J.), 386, 203–225. https://doi.org/10.1007/1-59745-430-3_7
 +
 +
4. Seidler, P., Boyer, D., Rodriguez, J., Sawaya, M., Cascio, D., Murray, K., Gonen, T., & Eisenberg, D. (2018). Structure-based inhibitors of tau aggregation. Nature chemistry, 10(2), 170. https://doi.org/10.1038/nchem.2889
 +
 +
5. 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.
  
 +
6. Stein, V., & Alexandrov, K. (2014). Protease-based synthetic sensing and signal amplification. Proceedings of the National Academy of Sciences, 111(45), 15934-15939
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4165021 parameters</partinfo>
 
<partinfo>BBa_K4165021 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 04:20, 14 October 2022


HtrA1 switch 1

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 1 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 TD28REV and WWW peptides which are 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, 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. The process of assembly of the whole switch was done accoding to both CAPRI and NCBI protocols.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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 with a score of 4 out of 6 according to our quality assessment code. 

                          Figure.1: This figure shows the Switch 1 top model by Pymol visualization.

Table 1: Quality assessment parameters of Switch 1 model.

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 4.23 1.2 99.3 0.7 0.384058 -0.7745

Switch construction Pipeline

1) Modelling

Since our Switch parts (HTRA1 binding peptide, TAU, and Beta-amyloid Binding peptide) do not have experimentally acquired structures, we modeled each one of them separately. 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 generated structures, we used the Swiss-Model tool, which gives an overall quality of any 3D structure (For more information, please check our 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 out of score 6. For more information: Programming club page code under the name of Modric..

4) Filtering

We take the top-ranked models from the previous steps that have either a score of 5 or 6

5) Docking

The top models of inhibitor and HTRA Binding Peptide are docked with HtrA1, and the top models of the clamps are docked with the Target protein, that is, in our case is Beta-amyloid (BBa_K4165004).

6) Ranking

The docking results are ranked according to the Delta free energy generated by PRODIGY. For more information please check our Docking page.

7) Top Models

The results from PRODIGY are ranked, and the top three models are chosen after the models are visualized to ensure that the proteins interact at the right designated domain to proceed with the next step. For more information please check our Docking page.

8) Alignment

Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex, the HtrA1-inhibitor complex, to check whether they bonded to the same site.

Figure 3. 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 between both C terminals, N terminals, C- and N- terminal, and N- and N- and C-terminals. <p style=" font-weight: bold; font-size:14px;">10) Assembly

After settling on the linkers' lengths, we will now 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 quality of any 3D structure (For more information, please check our Modeling 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, please proceed to our Programming club under the name of Modric.

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 6.33 1.7 94.89 0.73 -1.65604 -2.4253

14) Alignment

The docked structures are then aligned and compared to the basic parts, which are docked with the protein of interest (HtrA1). The structures with the least RMSD are chosen following the recommended range provided by CAPRI protocol.

RMSD Before Docking RMSD After Docking
1.41 1.85


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.

References

1. Goedert, M., & Spillantini, M. G. (2017). Propagation of Tau aggregates. Molecular Brain, 10. https://doi.org/10.1186/s13041-017-0298-7

2. Etienne, M. A., Edwin, N. J., Aucoin, J. P., Russo, P. S., McCarley, R. L., & Hammer, R. P. (2007). Beta-amyloid protein aggregation. Methods in molecular biology (Clifton, N.J.), 386, 203–225. https://doi.org/10.1007/1-59745-430-3_7

4. Seidler, P., Boyer, D., Rodriguez, J., Sawaya, M., Cascio, D., Murray, K., Gonen, T., & Eisenberg, D. (2018). Structure-based inhibitors of tau aggregation. Nature chemistry, 10(2), 170. https://doi.org/10.1038/nchem.2889

5. 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.

6. Stein, V., & Alexandrov, K. (2014). Protease-based synthetic sensing and signal amplification. Proceedings of the National Academy of Sciences, 111(45), 15934-15939