After identifying the role of Exotoxin A (ExoA) in the cytotoxicity of Pseudomonas aeruginosa, we sought to design and test protein mini-binders that would inhibit ExoA. The goal of our project was to develop a protein co-therapeutic that could serve as an adjunctive therapy alongside a regular antibiotic regimen in a Pseudomonas aeruginosa treatment plan.
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Computational Design of ExoA-Binding Proteins
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Design 1: Selecting An ExoA Binding Epitope for ExoA Functional Inhibition
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Design 2: Selection of Additional Hotspots for RFdiffusion
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Design 3: Introduction of Partial Diffusion for Increased Structural Diversity
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Design 4: Final Filter Using Additional Metrics
In-Vitro Assessment & Wet Lab
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Design 1: In vitro model of Exotoxin A toxicity
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Design 2: Editing Exotoxin A for lab uses
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Design 3: Miniproteins for binding and inhibition of Exotoxin A
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Design 4: Scheme for protein minibinder evaluation
References
[1] M. Michalska and P. Wolf, “Pseudomonas Exotoxin A: optimized by evolution for effective killing,” Frontiers in Microbiology, vol. 6, Sep. 2015. doi:10.3389/fmicb.2015.00963
[2] C. Guidi‐Rontani and R. J. Collier, “Exotoxin A of Pseudomonas Aeruginosa: Evidence that domain I functions in receptor binding,” Molecular Microbiology, vol. 1, no. 3, pp. 67–72, Nov. 1987. doi:10.1111/j.1365-2958.1987.tb00528.x
[3] Y. Jinno, V. K. Chaudhary, T. Kondo, S. Adhya, D. J. FitzGerald, and I. Pastan, "Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity.," Journal of Biological Chemistry, vol. 263, no. 26, pp. 13203–13207, 1988, doi: 10.1016/S0021-9258(18)37692-0
[4] G. J. Chaudry, R. B. Wilson, R. K. Draper, and R. C. Clowes, “A Dipeptide Insertion in Domain I of Exotoxin A that Impairs Receptor Binding,” Journal of Biological Chemistry, vol. 264, no. 25, pp. 15151–15156, Sep. 1989. doi:10.1016/s0021-9258(18)63824-4
[5] J. L. Watson et al., “De novo design of protein structure and function with RFdiffusion,” Nature, vol. 620, no. 7976, pp. 1089–1100, Jul. 2023. doi:10.1038/s41586-023-06415-8
[6] J. Dauparas et al., “Robust deep learning–based protein sequence design using ProteinMPNN,” Science, vol. 378, no. 6615, pp. 49–56, Oct. 2022. doi:10.1126/science.add2187
[7] J. Abramson et al., “Accurate structure prediction of biomolecular interactions with alphafold 3,” Nature, vol. 630, no. 8016, pp. 493–500, May 2024. doi:10.1038/s41586-024-07487-w
[8] “Confidence scores in Alphafold-Multimer,” Alphafold a practical guide, https://www.ebi.ac.uk/training/online/courses/alphafold/inputs-and-outputs/evaluating-alphafolds-predicted-structures-using-confidence-scores/confidence-scores-in-alphafold-multimer/ (accessed Oct. 6, 2025).
[9] R. N. Morgan, S. E. Saleh, H. A. Farrag, and K. M. Aboshanab, “New insights on Pseudomonas aeruginosa exotoxin A-based immunotoxins in targeted cancer therapeutic delivery,” Therapeutic Delivery, vol. 14, no. 1, pp. 31–60, Jan. 2023, doi: 10.4155/tde-2022-0055.
[11] ExoA catalytic mutant - M. Lukac, G. B. Pier, and R. J. Collier, “Toxoid of Pseudomonas aeruginosa exotoxin A generated by deletion of an active-site residue,” Infection and Immunity, vol. 56, no. 12, pp. 3095–3098, Dec. 1988, doi: 10.1128/iai.56.12.3095-3098.1988.