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Vascular Endothelial Growth Factor Receptor 2 (VEGF-R2)

Part Description

It is vascular endothelial growth factor receptor which contain 3 members contain similar structures and their external parts are made up entirely of repeating segments that resemble parts of antibodies (immunoglobulin homology repeats) and they have a critical role in the formation of blood vessels and lymphatic vessels.

Usage

This is the extracellular domain of the second chain dCas9-synRTK receptor which respond specifically to vascular endothelial growth factor (VEGF) which is a specific substance for wounds so when activated it transduce the signal causing dimerization of the two receptor chains and thus help in the control of transcription and prevent auto activation.

this figure illustrates the structure of the extracellular domain of our receptor's second chain .

Literature Characterization

In this study, a group of 34 angiosarcomas were examined using immunohistochemistry technique which allowed to assess the levels of proteins involved in blood vessel growth, including vascular endothelial growth factors (VEGF-A and VEGF-C) and their corresponding receptors (VEGFR-1, VEGFR-2, and VEGFR-3).

VEGF-A was expressed by 32/34 (94%), VEGF-C by 4/34 (12%), VEGFR-1 by 32/34 (94%), VEGFR-2 by 22/34 (65%), and VEGFR-3 by 27/34 (79%) .

The figure shows immunohistochemical staining of proteins associated with blood vessel growth in angiosarcoma scalp tissue. Panel A reveals strong positivity for VEGF-A in tumor cells. Panels B and C demonstrate consistent positivity for VEGFR-1 and VEGFR-3, respectively, in the lining of tumor cells. Panel D shows VEGF-C expression within the angiosarcoma, surrounded by lighter areas containing lymphocyte immune cells.

Software Characterization

To choose the best external domain among the VEGFR2, we started performing homology modelling to predict our external domains’ three-dimensional structure through Swiss model online tool that uses template homology modelling method to predict the 3D structure.

(a)Ramachandran plot

(b)VEGFR2 3d structures

this figure The Ramachandran plot shows that 93.36% of the VEGFR2 amino acid residues lie in a favorable region, indicating a stable protein structure. On the other hand, 0.89% of amino acid residues are considered as outliers. .

We also utilized Alpha fold3 server that uses de-novo homology modelling method to predict the 3D structure and to align this structure with the similar experimented structures.

The alignment plot of VEGFR2 shows high intensity along the diagonal line indicating high similarity between the 3D structure and the experimental structures. .

Then, In order to measure the binding stability between VEGFR2 and their ligand VEGFA, which is the main function of the external domain, we performed molecular docking using alpha fold 3 server.

The alignment plot shows positive alignment between the 3D structure’s amino acids and the experimental structures in the diagonal line indicating good protein structure. Additionally, they scored binding stability (ΔG) of -9.2 kcal mol-1.

Then, we performed directed evolution using EV couplings to detect the highest and the lowest epistatic fitness of the VEGFR1 to be used in our project to choose the best mutant variant of the VEGFR2

This heatmap shows VEGFR-2 mutational landscape which is generated by EVcouplings software. It shows that the mutant variant associated with the highest epistatic fitness was (Y 418 N), meanwhile the highest independent score was also related to the (Y 418 N).

Reference

Itakura, E., Yamamoto, H., Oda, Y., & Tsuneyoshi, M. (2008). Detection and characterization of vascular endothelial growth factors and their receptors in a series of angiosarcomas. Journal of surgical oncology, 97(1), 74-81.‏

Sequence and Features