Tuning flavin environment to detect and control light-induced conformational switching in Drosophila cryptochrome

Abstract

Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools.

Cryptochromes: Photochemical and structural insight into magnetoreception

“…Specifically, Type I CRYs exist in an FAD ox ground state, but do not generate FADH • , rather blue‐light illumination leads to FAD •‐ without further protonation or photochemical reduction (Table 1). 12,81 Analysis of redox potentials of Type I CRYs confirm they should exist in the FAD ox state under cellular conditions, and limited proteolysis studies confirm that FAD •− formation is both necessary and sufficient for downstream signaling 82–84 . Thus, Type I CRYs should function through an M1 mechanism in magnetoreception.…”

“…12,81 Analysis of redox potentials of Type I CRYs confirm they should exist in the FAD ox state under cellular conditions, and limited proteolysis studies confirm that FAD •À formation is both necessary and sufficient for downstream signaling. [82][83][84] Thus, Type I CRYs should function through an M1 mechanism in magnetoreception.…”

“…Both C416D and C416N variants are capable of generating some FADH • , but with low yield. 84 Structural investigations indicate that solvent access to the N5 position is also necessary for tuning photochemical outcomes. Robust generation of the FADH • state requires both L405E and C416N variants, where introduction of the L405E variant found in plant CRYs and Type IV CRYs leads to selection of the neutral semiquinone 84 (Figure 4 (a)).…”

“…84 Structural investigations indicate that solvent access to the N5 position is also necessary for tuning photochemical outcomes. Robust generation of the FADH • state requires both L405E and C416N variants, where introduction of the L405E variant found in plant CRYs and Type IV CRYs leads to selection of the neutral semiquinone 84 (Figure 4 (a)). The ability to tune photochemical outcomes in CRY systems has afforded researchers a valuable tool instrumental in deciphering the ground and signaling states gating signal transduction in CRY proteins.…”

“…Namely, H378R/K variants stabilize dCRY against light-dependent degradation but retain activity against TIM. 84 Currently, no structures of dCRY protein complexes exist, however some insight may be garnered from structural and biophysical characterization of Type II CRYs, where the CTT gates competitive interactions at the primary and secondary pocket to gate selection of CRY protein-protein interactions. 110…”