Protein Surface Site Determines the Evolutionary Accessibility of Allosteric Regulation

Domain recombination is a major source of new allosteric regulation in both evolved and engineered proteins. However, the sequence and structural features that govern where new allostery may emerge remain poorly understood. Here, we test the hypothesis that the evolutionary accessibility of allosteric regulation following domain insertion is constrained by local surface context, specifically association with pre-existing cooperative networks known as protein sectors. We began with two synthetic domain fusions wherein the Avena sativa light-oxygen-voltage (LOV2) domain was inserted into Escherichia coli dihydrofolate reductase (DHFR) at either a sector connected or non-sector connected surface. The insertion sites are only separated by five residues and both DHFR enzymes retain similar catalytic activity, yet the sector connected version exhibits a light-dependent allosteric phenotype, while the non-sector connected version does not. Using deep mutational scanning, we measured the effect of nearly all single point mutations on allostery in each chimera. The sector-connected DL121 was significantly more evolvable, possessing numerous allostery-tuning single mutants. In contrast, DL116 lacked statistically significant mutants that introduce allosteric regulation, suggesting the protein surface used by DL116 may be an evolutionary "dead end" for a regulatory phenotype. Surprisingly, DL116 did not show cooperative unfolding at temperatures up to 80 {degrees}C, suggesting that enhanced protein stability does not promote the evolvability of allosteric regulation as it does with other phenotypes. Together, our findings show that protein surface context influences the mutational pathways available for allosteric regulation, consistent with the view that sector-connected surface sites harbor a latent capacity for allostery while other locations are more evolutionarily inert.