Structurally Distributed Surface Sites Tune Allosteric Regulation

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.

Global mapping of the energetic and allosteric landscapes of protein binding domains

“…In particular, PPIs represent a very large but difficult to inhibit therapeutic target space and the systematic identification of allosteric sites may open up opportunities for inhibiting targets currently considered ‘undruggable’ 14 . Moreover, systematic maps of spatial information transfer in proteins—and selection experiments that identify modifiers of this transfer 16,21,22 —should provide fundamental insights into the mechanisms that underlie allostery, elucidate how it evolves, and quantify the extent to which allostery varies within and between protein families.…”

“…An example is a cryptic allosteric pocket in the oncoprotein KRAS targeted by the first clinically approved RAS inhibitor 14 . Moreover, domain-insertion and mutagenesis also suggest quite extensive long-range communication in protein interaction domains 15 , enzymes [16][17][18][19][20] , transcription factors [21][22][23] and receptors 24,25 . Physical interactions between proteins are critical to most biological processes and, with >50,000 physical interactions between human proteins now identified and a total estimated interactome of >500,000 edges 6 , protein-protein interactions (PPIs) represent a vast and largely untapped therapeutic target space 6 . However, protein interaction interfaces are considered difficult drug targets 26 .…”

“…An example is a cryptic allosteric pocket in the oncoprotein KRAS targeted by the first clinically approved RAS inhibitor 14 . Moreover, domain-insertion and mutagenesis also suggest quite extensive long-range communication in protein interaction domains 15 , enzymes 16–20 , transcription factors 21–23 and receptors 24,25 .…”

Structurally distributed surface sites tune allosteric regulation

“…We describe each step below; all code was implemented in Bash shell scripting or Python 3.6.4. All analysis codes have been made available as a series of python 3 Jupyter Notebooks on github ( https://github.com/reynoldsk/allostery-in-dhfr ; McCormick et al, 2021 ; copy archived at swh:1:rev:dd8ee13f775f8b08548d64868f15e46583cbf543 ).…”