Thomas Truong (U): VVD and ENV Equivalent Mutant Characterization

Light-Oxygen-Voltage (LOV) domains, present in all kingdoms of life, facilitate regulation of light dependent events in organisms by transducing light input into physiological signals. Examination of plant and fungal circadian networks has revealed that signaling mechanisms differ even within homologs of closely related species. Structural and computational studies of LOV-allostery have identified key signaling “hot-spots” that allow modification of the direction and amplitude of signal output. One key regulatory site was identified in Plant LOV domain proteins ZTL and FKF1, in which the amino acid residue at position 46 differentiates ZTL and FKF1 signaling mechanisms. Notably, the residue at this position differentiates FKF1-based signaling in monocots and dicots (Ala and Ser, respectively). An analogous residue substitution is observed in the LOV domain photoreceptors of two closely related filamentous fungi, Neurospora crassa VIVID and Trichoderma reesei ENVOY, where divergent signaling mechanisms gate adaptation to oxidative stress. Herein, we sought to test whether residue identity at the position equivalent to ZTL G46 (VIVID A72 and ENVOY S99) alters signal transduction in VIVID and ENVOY. To propagate signal transduction of blue-light, wildtype VVD forms a rapidly dissociating dimer whereas wildtype ENV requires oxidative conditions to form an irreversible disulfide dimer. Size exclusion chromatography (SEC) characterization revealed VVD A72S exhibits reduced dimerization capability and dimerizes at high concentrations only. Furthermore, ENV S99A dimerized under reducing conditions with a similar concentration-dependent increase in dimeric fraction. This altered dimerization capacity demonstrates evolutionary selection of G46 equivalent residues in differentiating LOV-domain photoreceptors and their signal transduction mechanisms.