RT Journal Article SR Electronic T1 Structurally Distributed Surface Sites Tune Allosteric Regulation JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.03.11.435042 DO 10.1101/2021.03.11.435042 A1 McCormick, James W. A1 Russo, Marielle A.X. A1 Thompson, Samuel A1 Blevins, Aubrie A1 Reynolds, Kimberly A. YR 2021 UL http://biorxiv.org/content/early/2021/03/12/2021.03.11.435042.abstract AB 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 StatementThe authors have declared no competing interest.