Structurally distributed surface sites tune allosteric regulation
- University of Texas Southwestern Medical Center, United States
- Stanford University, United States
Abstract
Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mutations that tune allostery. Are these mutations few or abundant, structurally localized or distributed? To examine this, we conducted saturation mutagenesis of a synthetic allosteric switch in which Dihydrofolate reductase (DHFR) is regulated by a blue-light sensitive LOV2 domain. Using a high-throughput assay wherein DHFR catalytic activity is coupled to E. coli growth, we assessed the impact of 1548 viable DHFR single mutations on allostery. Despite most mutations being deleterious to activity, fewer than 5% of mutations had a statistically significant influence on allostery. Most allostery disrupting mutations were proximal to the LOV2 insertion site. In contrast, allostery enhancing mutations were structurally distributed and enriched on the protein surface. Combining several allostery enhancing mutations yielded near-additive improvements to dynamic range. Our results indicate a path towards optimizing allosteric function through variation at surface sites.
Data availability
Sequencing data (resulting from amplicon sequencing) have been deposited in the NCBI SRA under BioProject: PRJNA706683All analysis codes have been made available as a series of python 3 Jupyter Notebooks on github: https://github.com/reynoldsk/allostery-in-dhfr
Article and author information
Author details
Kimberly A Reynolds
Funding
National Science Foundation (CAREER Award,1942354)
- Kimberly A Reynolds
Gordon and Betty Moore Foundation (Data Driven Discovery Initiative,GBMF4557)
- Kimberly A Reynolds
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, McCormick et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Structurally distributed surface sites tune allosteric regulation
eLife 10:e68346.
https://doi.org/10.7554/eLife.68346
