Local disorder is associated with enhanced catalysis in a nascent photoswitch

Domain insertion is a common strategy for introducing allosteric regulation in both engineered and evolved systems. In this approach, an "input" domain is covalently fused to an "output" domain with the goal of conferring new regulation. In prior work, we found that insertion of the LOV2 domain at evolutionarily conserved allosteric "hot spots" on the metabolic enzyme Dihydrofolate Reductase (DHFR) could confer modest light regulation of enzymatic activity. However, it was not clear if the newly established regulation was achieved by interdomain allosteric conformational coupling, or if it represented a "simpler" mechanism like coupling of LOV2 light activation to global folding stability of DHFR or light-dependent steric occlusion of the DHFR active site. To better understand how these newly formed domain fusions harness light-inducible disorder in LOV2 for allosteric activation, we biochemically characterized a representative synthetic fusion. We observed that LOV2 photoactivation simultaneously: (1) thermally destabilized the fusion and (2) lowered the DHFR catalytic transition free energy of the lit state relative to the dark state. Light-induced NMR chemical shift changes indicated that photochemically-initiated conformational changes propagated from LOV2 to the active site of DHFR. Moreover, ligand binding at DHFR modified LOV2 chemical shifts, demonstrating bidirectional coupling between domains. Examination of select allostery-tuning mutations found a modest negative correlation between the light-induced change in thermal stability and catalytic activity, suggesting an activity-stability tradeoff. Together our data indicate that a domain fusion event can realize localized conformational coupling between active sites even in the absence of extensive evolutionary optimization.