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 James W. McCormick
A1 Marielle A.X. Russo
A1 Samuel Thompson
A1 Aubrie Blevins
A1 Kimberly A. Reynolds
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.