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.

Fig. 1. Structure of dCRY.

a The C-terminal tail (CTT) of dCRY (red) binds into the pocket containing FAD (yellow). b C416 (green) interacts with the FAD N⁵ to disfavor protonation, whereas L405 blocks the solvent from accessing the flavin. The N416 and E405 substitutions (tan) favor NSQ formation. The L405E/C416N replacements are located at the distal end from the CTT with the FAD group sandwiched in-between the two sites. c H378 (green) bridges the flavin (magnesium shown as green spheres) to the FFW motif of the CTT.

Fig. 2. An NSQ-forming dCRY variant.

a Structural and sequence alignment of dCRY (gray) and aCRY (tan), key residues that affect the nature of the radical formed are highlighted in red. UV–vis spectroscopy (b) and Q-band ENDOR spectra (c) of dCRY WT (red) and dCRY L405E/C416N (blue) variant. Frequencies ±1.5 MHz around the central Larmor frequency correspond to solvent protons as well as protons on the isoalloxazine ring (H³, H⁷, and H⁹). The dCRY L405E/C416N variant has hyperfine coupling values at 5.0, 6.8, 10.8, and 25.3 MHz corresponding to the H⁶, H^8α, H^1’, and H⁵ protons, respectively. The broad hyperfine coupling observed at 25.3 MHz for H⁵ (inset) is characteristic of an NSQ radical. In comparison, the H8α and H6 protons of WT dCRY have ¹H frequencies at 10.5 and 5.3 MHz, respectively. The larger hyperfine value of 10.5 MHz for H^8α in WT dCRY compared to 6.8 MHz in dCRY L405E/C416N is typical of an ASQ.

Fig. 3. Site-specific C-terminal protein labeling using sortase A.

a Sortase A catalyzes the ligation of a polyglycine probe (GGGGC) to an LPGTG recognition motif. For analysis using ESR spectroscopy, the cysteine of the glycine probe was conjugated to a nitroxide radical. b cw-ESR spectra of (top) labeled GGGGC peptide, (bottom) C terminally labeled A. thaliana iLOV. c Photoreduction of iLOV allows distance measurements between the flavin NSQ and sortylated nitroxide radical. DEER measurements (time domain, left) for C-terminally labeled iLOV indicated a broad distance distribution (right).

Fig. 4. Flavin-nitroxide DEER spectroscopy of irradiated dCRY.

4P-DEER time-domain trace (a) and pulse dipolar distance distribution (b) obtained between active site flavin and nitroxide radicals on CTT of dCRY variant. The distance distribution is shorter and narrower for the NSQ forming dCRY L405E/C416N (blue) variant when compared to the WT protein (pink) after light exposure. The error values (${\chi }_v^2$) for the distance reconstruction are close to 1, indicating a good fit; the error bar on the distance values are below 0.2 Å. c The crystal structure of dCRY-WT-SORTC-SL in the dark state shows that the active site FAD and the sulfhydryl group on the GGGGC linker are separated by ~31–32 Å (depending on the FAD atom chosen). Magnesium atoms are shown as green spheres.

Fig. 5. H378 substitutions affect CTT conformation in dark and in light.

PDS distance distributions between the flavin and nitroxide radicals for the various H378 dCRY variants. Each spectrum was fit to a combination of docked (L405E/C416N distance distribution) and undocked (WT distance distribution) states. The H378R/K/N variants show a mixture of docked and undocked states even in the dark state proxy of L405E/C416N (top panels).