Signal transduction in light–oxygen–voltage receptors lacking the adduct-forming cysteine residue

Abstract

Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.

Optogenetic Control by Pulsed Illumination

“…Next, we assessed the response to intensity and pulse frequency of illumination in pDusk and pDawn systems harboring YF1 variants that lacked the conserved adduct‐forming Cys62 inside the LOV photosensor (Figures and S2). We previously showed that such cysteine‐devoid LOV receptors could still retain light sensitivity and downstream signal transduction because, in the absence of the conserved cysteine, blue‐light absorption promotes reduction of the flavin chromophore to the neutral semiquinone state (NSQ) . Notably, formation of the NSQ entails protonation of the flavin N5 atom, which is key to triggering downstream responses.…”

“…Concomitant with thioether formation, the N5 atom of the flavin chromophore is protonated, which triggers hydrogen‐bond rearrangements throughout the LOV photosensor. Studies on cysteine‐devoid variants of LOV receptors revealed that N5 protonation was both necessary and sufficient for downstream signal propagation . The stability of the thioether bond in cysteine‐containing LOV receptors is strongly governed by temperature, solvent composition, and molecular environment of the flavin chromophore .…”

Glutamine Amide Flip Elicits Long Distance Allosteric Responses in the LOV Protein Vivid

“…In a previous study a VVD variant in which the active site Cys has been changed to Ala was also found to signal after photoreduction of the flavin to the neutral semiquinone state, which also has N5 protonated. 19 In our simulations the Cys-less variant also gives rise to the Gln conformational switch and thus points to a similar conformational activation mechanism in the Cys-less variant. Finally, free energy simulations indicate that in the light state the barrier for Gln182 flipping is reduced and the flipped conformation is substantially stabilized.…”

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY

“…1, A and B). Such behavior suggests that the oxidation potential of the FMN cofactor may reside within physiologically relevant ranges as has been observed previously in LOV protein variants (18,34) and has recently been shown of being competent for signal transduction (35). Thus, ENV1 may employ the semiquinone in dark state signaling.…”