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Structurally Distributed Surface Sites Tune Allosteric Regulation

View ORCID ProfileJames W. McCormick, Marielle A.X. Russo, Samuel Thompson, Aubrie Blevins, View ORCID ProfileKimberly A. Reynolds
doi: https://doi.org/10.1101/2021.03.11.435042
James W. McCormick
1The Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, USA, 75390
2Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, USA, 75390
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  • ORCID record for James W. McCormick
Marielle A.X. Russo
1The Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, USA, 75390
2Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, USA, 75390
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Samuel Thompson
3Department of Bioengineering, Stanford University, Stanford, CA 94305
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Aubrie Blevins
1The Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, USA, 75390
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Kimberly A. Reynolds
1The Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, USA, 75390
2Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, USA, 75390
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  • ORCID record for Kimberly A. Reynolds
  • For correspondence: kimberly.reynolds@utsouthwestern.edu
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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.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://github.com/reynoldsk/allostery-in-dhfr

  • https://www.ncbi.nlm.nih.gov/bioproject/PRJNA706683

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted March 12, 2021.
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Structurally Distributed Surface Sites Tune Allosteric Regulation
James W. McCormick, Marielle A.X. Russo, Samuel Thompson, Aubrie Blevins, Kimberly A. Reynolds
bioRxiv 2021.03.11.435042; doi: https://doi.org/10.1101/2021.03.11.435042
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Structurally Distributed Surface Sites Tune Allosteric Regulation
James W. McCormick, Marielle A.X. Russo, Samuel Thompson, Aubrie Blevins, Kimberly A. Reynolds
bioRxiv 2021.03.11.435042; doi: https://doi.org/10.1101/2021.03.11.435042

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