Latest revision as of 15:07, 1 October 2024

VEGF121(R105S, P106Y) Enhancing the Formation of VEGF-VEGFR Complex

A mutation of VEGF121 on two amino acids enhancing the formation of VEGF-VEGFR complex by the formation of an additional hydrogen bond

Sequence and Features

This part is improved from BBa_K229800, and mutated with stronger receptor binding capacity and the ability to promote cell migration/wound healing.

We have carefully redesigned the existing VEGF121 part by introducing targeted mutations at two key residues, R105S and P106Y, aiming to enhance its binding affinity to VEGFR and improve its biological function. Our efforts are demonstrated through rigorous experimental validation, including a scratch assay with MC3T3-E1 cells, which shows that the mutant significantly enhances cell migration and wound healing compared to the wild-type VEGF121. Additionally, we have meticulously documented our findings, following the Best New Improved Part award requirements. This includes detailed experimental protocols, quantitative results, and clear comparisons between the mutant and wild-type proteins under identical conditions. Our thorough documentation on the Part's Registry Main Page ensures that the new part is well-supported by experimental data, showcasing its superior performance.

1 Targeted Mutagenesis of VEGF to Enhance Receptor Binding and Boost Angiogenesis

In clinical settings, rapid angiogenesis is essential, but achieving this requires high concentrations of VEGF, which can be costly and potentially trigger immune reactions and side effects due to excessive cell factor levels. Therefore, there is a pressing need for a molecule that can promote effective blood vessel growth at lower concentrations. To address this, we aim to engineer VEGFA through rational design, enhancing its receptor-binding efficiency. This modification is expected to result in better angiogenic outcomes with reduced cell factor concentrations, improving therapeutic efficacy.

The activity of wild type VEGF is currently limited, which presents a challenge because rapid stimulation of angiogenesis is crucial in therapeutic contexts. On a molecular level, increasing the binding affinity between VEGF and its receptor (VEGFR) could significantly enhance the speed and effectiveness of this process. By performing targeted mutagenesis on VEGFA, we aim to improve receptor interaction, thereby boosting VEGF’s bioactivity. This modification would lead to more efficient promotion of vascular growth, an essential factor in tissue regeneration and repair applications such as HEK293 cell migration studies.

2 Investigating the mechanism of interaction between VEGF and VEGFR

Thus, we began to study the interaction between VEGF and its receptor. Using VEGF121 as a model, we focused on its interaction with the extracellular domain of VEGFR (PDB: 4CKV). We applied AlphaFold3 to analyze the interaction mechanism between VEGF121 and VEGFR, allowing us to explore the structural details of their binding. This approach aims to provide insights into how rationally designed mutations in VEGF can enhance receptor binding and angiogenesis at lower cell factor concentrations.

Fig2.1 Wild-type VEGF (cyan) interacts with VEGFR (extracellular domain, green), with yellow dashed lines representing hydrogen bonds

Fig2.2 All schematic diagram of the interaction between wild-type VEGF (cyan) and VEGFR (extracellular domain, green). The yellow dotted lines represent hydrogen bonds.

With only three hydrogen bonds identified between VEGF and VEGFR (K16-E10, D63-R93, Y25-L73), the relatively weak interaction is further confirmed by the AutoDock Vina score of -6.3, indicating suboptimal binding energy. Such a low binding score suggests that the current VEGF structure has limited capacity to strongly engage with its receptor. Enhancing VEGF’s binding affinity is crucial for improving its biological function, particularly for therapeutic purposes like stimulating angiogenesis. Optimizing these interactions through mutagenesis can strengthen the receptor engagement and boost angiogenic activity, making VEGF more effective in promoting rapid blood vessel formation and tissue repair. By strategically increasing hydrogen bond numbers or improving molecular stability, we can potentially create a more efficient and potent version of VEGF for clinical applications.

We ultimately decided to increase the number of hydrogen bonds rather than focusing on thermal stability for several reasons. First, enhancing hydrogen bonding directly improves the binding affinity between VEGF and VEGFR, which is crucial for stronger receptor interactions and better signaling. In contrast, improving thermal stability may only extend the protein's half-life but won't necessarily enhance its receptor-binding efficiency. Prolonged half-life may cause cytokines to spread to other sites, causing side effects. Since our goal is to boost biological activity, increasing hydrogen bond interactions provides a more direct and effective approach for improving VEGF's functionality.

3 Mutants

From the perspective of hydrogen bond formation, enhancing VEGF's interaction with VEGFR can be achieved by targeting amino acids in the interface region that currently cannot form hydrogen bonds. Mutations should aim to introduce amino acids with hydrogen-bonding capabilities.

Typically, residues like proline (P), which has a rigid cyclic structure that cannot form hydrogen bonds, can be mutated to tyrosine (Y) or serine (S), which possess hydroxyl groups that actively participate in hydrogen bonding. Other examples include mutating alanine (A) to glutamine (Q) or asparagine (N) to introduce polar side chains capable of forming hydrogen bonds.

By strategically selecting and mutating such residues in the binding region, we can significantly improve the VEGF-VEGFR interaction, leading to stronger receptor engagement and enhanced biological activity.

Fig3.1 Mutations lead to increased hydrogen bonding

Based on the results from multiple AlphaFold3 simulations, we found that mutating P106 to Y while also mutating R105 to S can increase the number of hydrogen bonds without compromising the stability of the structure. If only P106 is mutated to Y, the large steric hindrance from R105, which is located at the turn, causes structural instability. By mutating R105 to a smaller amino acid like S, we can maintain structural integrity while improving the interaction through additional hydrogen bonds. After the R105S and P106Y mutations, Y106 forms a hydrogen bond with E19 of VEGFR, further enhancing the binding affinity between VEGF and VEGFR. This newly introduced hydrogen bond provides a more stable interaction that could improve the biological effectiveness of VEGF in therapeutic applications.

Fig3.2 VEGF (wild type & R105S and P106Y mutation, cyan) interacts with VEGFR (extracellular domain, green), with yellow dashed lines representing hydrogen bonds

4 Experimental verification

The scratch assay, also known as the wound healing assay, is a widely used method for studying cell migration. It simulates wound healing by creating a "scratch" in a cell monolayer, and the ability of cells to move into this gap is tracked over time.

To assess the wound-healing capabilities of our VEGF mutants (R105S and P106Y), we will conduct a scratch assay using MC3T3-E1 cells. Cells will be cultured in a 6-well plate until 80-90% confluency, followed by a scratch using a pipette tip. Afterward, serum-free medium containing 50 ng/mL of either wild-type or mutant VEGF will be added. Images of the scratch will be taken at 24 hours to monitor wound closure. We will compare the wound closure rates between the wild-type and mutant VEGF to evaluate enhanced cell migration.

Fig4.1 VEGF121 Mutant (R105S, P106Y) Enhances Wound Healing and Cell Migration

The data from the scratch assay show a significant improvement in both wound healing percentage and cell migration velocity for the VEGF121(R105S, P106Y) mutant compared to the wild-type VEGF121. The left graph demonstrates that the wound healing percentage for the mutant is significantly higher (p < 0.01) than for the wild-type. Similarly, the right graph indicates that the cell migration velocity is also significantly increased in the mutant (p < 0.05). These results suggest that the R105S and P106Y mutations enhance VEGF's ability to promote cell migration and wound healing.

The results of the scratch assay demonstrate that the VEGF121 mutant (R105S, P106Y) significantly outperforms the wild-type VEGF121 in both wound healing percentage and cell migration velocity. The increased wound healing percentage indicates that the mutant VEGF enhances cell migration and wound closure more effectively than the wild-type. The higher cell migration velocity in the mutant further supports this finding, suggesting that the mutations enhance the interaction between VEGF and VEGFR, likely due to increased hydrogen bonding and receptor binding affinity. This improvement could have valuable implications for therapeutic applications where rapid angiogenesis and tissue repair are needed.