Full and partial agonism of a designed enzyme switch
Open accessAbstract
Chemical biology has long sought to build protein switches for use in molecular diagnostics, imaging, and synthetic biology. The overarching challenge for any type of engineered protein switch is the ability to respond in a selective and predictable manner that caters to the specific environments and time scales needed for the application at hand. We previously described a general method to design switchable proteins, called "chemical rescue of structure", that builds de novo allosteric control sites directly into a protein's functional domain. This approach entails first carving out a buried cavity in a protein via mutation, such that the protein's structure is disrupted and activity is lost. An exogenous ligand is subsequently added to substitute for the atoms that were removed by mutation, restoring the protein's structure and thus its activity. Here, we begin to ask what principles dictate such switches' response to different activating ligands. Using a redesigned β-glycosidase enzyme as our model system, we find that the designed effector site is quite malleable and can accommodate both larger and smaller ligands, but that optimal rescue comes only from a ligand that perfectly replaces the deleted atoms. Guided by these principles, we then altered the shape of this cavity by using different cavity-forming mutations, and predicted different ligands that would better complement these new cavities. These findings demonstrate how the protein switch's response can be tuned via small changes to the ligand with respect to the binding cavity, and ultimately enabled us to design an improved switch. We anticipate that these insights will help enable the design of future systems that tune other aspects of protein activity, whereby, like evolved protein receptors, remolding the effector site can also adjust additional outputs such as substrate selectivity and activation of downstream signaling pathways.
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Addition of indole in allosteric cases serves not to revert a discrete conformational change, but as an allosteric ligand that rescues activity by inducing the protein to refold to its original conformation.
“Addition of indole in these allosteric cases served not to revert a discrete conformational change, but rather as an allosteric ligand that rescues activity by inducing the protein to refold to its original conformation (Budiardjo et al., 2016)”
From Allostery in Its Many Disguises: From Theory to Applications (2019) · cites this paper as Budiardjo et al., 2016
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Allostery in Its Many Disguises: From Theory to Applications
Wodak, S. J., Paci, E., Dokholyan, N. V., Berezovsky, I. N., Horovitz, A., Li, J., Hilser, V. J., Bahar, I., Karanicolas, J., Stock, G., et al. (2019) · Structure · doi:10.1016/j.str.2019.01.003 · Cited by 404 · see citing works
Self-citation
- Authors
- Budiardjo, S. J., Licknack, T. J., Cory, M. B., Kapros, D., Roy, A., Lovell, S., Douglas, J., & Karanicolas, J.
- Affiliations
- University of Kansas (US)
- Corresponding author
- Karanicolas, John
- Venue
- ACS Synthetic Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2016-07-22
- Volume
- 5
- Issue
- 12
- Pages
- 1475-1484
- ISSN
- 2161-5063 (ISSN-L)
- PMC
- PMC5161622
- OpenAlex
- W2473526301
- Cited by
- 13
- References
- 43
- Citations by year
- 2026: 4 · 2022: 1 · 2020: 2 · 2019: 4 · 2018: 1 · 2017: 1
- FWCI
- 1.04
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- ACS Synthetic Biology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Science Foundation (ACI-1053575)
- U.S. Department of Health and Human Services (5P20RR017708, 2R25GM078441, 5P30GM110761, 8P20GM103420)
- Alfred P. Sloan Foundation
- Arnold and Mabel Beckman Foundation
- Human Frontier Science Program
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Keywords
- protein switches, allosteric regulation, β-glycosidase, ligand-binding cavity, ligand specificity, enzyme activation, de novo protein design
- MeSH terms
- Allosteric Site, Binding Sites, Glucosidases, Ligands, Models, Molecular, Mutation, Magnetic Resonance Spectroscopy, Protein Binding, Protein Conformation, Proteins, Structure-Activity Relationship, Protein Engineering
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