Part:BBa_K5036000
Vascular Endothelial Growth Factor Receptor 1 (VEGF-R1)
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 first chain dCas9(C)-TF-synVEGFR1 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 first chain .
Dry lab Characterization
We performed 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)VEGFR1 3d structures
This Ramachandran plot shows that 93.44% of amino acid residues lie in a favorable region, indicating a well-formed protein.However, there are about 1.80% amino acid residues 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.
This alignment plot illustrates darker green color along the diagonal line, indicating lower expected position errors between the 3D structure and the experimental structures. .
Then, In order to measure the binding stability between VEGFR1 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 indicates a positive diagonal alignment between the 3D structure and the experimental structures which reflect favorable protein structure. Additionally, they scored binding stability (ΔG) of -8.6 kcal mol-1.
Then, we performed molecular dynamics for VEGFR1-VEGFA complex to measure its stability in normal physiological conditions
Being simulated in normal physiological conditions by amber notebook, VEGFR-1 shows an initial rise in RMSD from 2Å to 6Å which indicates changes in surrounding environment and conditions. After that, a state of fluctuation and deviation , respectively, indicating a stable protein with deviation less than 5 Å .
Then, we performed directed evolution using EV couplings to detect the highest epistatic fitness and independent score of the VEGFR1 to be used in our project to choose the best mutant variant of the VEGFR1
This heatmap shows VEGFR-1 mutational landscape which is generated by EVcouplings software. It shows that the mutant variant associated with the highest epistatic fitness was (M 356 V), meanwhile the highest independent score was also related to the (M 356 V) .
Characterization by Mathematical Modeling
The model provides the interaction kinetics of VEGFR1 external domain upon binding of VEGF to it , the result shows satisfactory binding state and stability based on parametric values from literature.
Graph (1). illustrates the decreasing of VEGFR2 (Red line) upon binding of VEGF (A) to form VEGF-VEGFR2 complex (R2A) (Yellow line) which increases till its binding to VEGFR1 (R1), so (R1) also decreases after will ( Black line). To finally form a binding state of fitted ligand receptor complex (R2AR1) (Blue line) .
Experimental Characterization
we digested dCas9(C)_NLS-Syn-VEGFR-1 grafetd with VEGFR-1 and performed gel electrophoresis to detect the digested fragment.
This figure illustrates the digested dCas9(C)_NLS-Syn-VEGFR-1 prior to insert ligation .
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.
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.
White C, Rottschäfer V, Bridge LJ. Insights into the dynamics of ligand-induced dimerisation via mathematical modelling and analysis. J Theor Biol. 2022 Apr 7;538:110996. doi: 10.1016/j.jtbi.2021.110996. Epub 2022 Jan 24. PMID: 35085533. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1555
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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