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

Siddarth Chandrasekaran,Connor M Schneps,Cristina C Deoliveira,Abir Ganguly,Changfan Lin,Brian R Crane,Robert Dunleavy

doi:10.1038/s42003-021-01766-2

Siddarth Chandrasekaran, Connor M Schneps + Show 5 more

Open Access

https://doi.org/10.1038/s42003-021-01766-2

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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.

Highlights

Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM)
A typical nitroxide moiety targeted to the dCRY C-terminal tail (CTT) can serve as the first spin, whereas, in principle, the flavin SQ can serve as the second
Plant orthologs of dCRY, such as the Arabidopsis thaliana cryptochrome 1 (AtCRY1) or Chlamydomonas reinhardtii animal-like cryptochrome, as well as the ClCRY4 protein from Columba livia, either photoreduce to the neutral SQ (NSQ) or naturally bind the NSQ in the dark[34,35,36]

Summary

Introduction

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). Blue light photoreduces the oxidized flavin cofactor of dCRY to the anionic semiquinone (ASQ) state[12,13,14,15], which induces a conformational change in the CTT that promotes binding to TIM15–18. Alterations to a conserved tetrad of tryptophan residues that supply electrons to the photoexcited flavin[24,25] affect dCRY photoreduction rates, dCRY light stability, and TIM degradation activity[26]. The dark-state structure of dCRY shows that the CTT binds to the FAD pocket through a conserved FFW motif (F534, F535, and W536), which contributes to dCRY stability[6,7,15,30]. Control of the CTT response would be advantageous for the development of optogenetic tools

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