Capturing structural intermediates in an animal-like cryptochrome photoreceptor by time-resolved crystallography

Animal-like cryptochromes are photoreceptors that control circadian rhythm and signaling in many eukaryotes. Transient photoreduction of the cryptochrome flavin chromophore initiated signaling via a poorly understood mechanism. By serial femtosecond crystallography (SFX), we show that the photoreduction mechanism of Chlamydomonas reinhardtii cryptochrome involves three loci [carboxyl-terminal region, a transient protonation pathway, and flavin adenine dinucleotide (FAD)-binding site] acting in unison to accomplish three effects: radical pair stabilization, protonation of FAD radical, and formation of the signaling state. Using 19 time-resolved SFX snapshots between 10 nanoseconds and 233 milliseconds, we found that light-driven FAD^•-/tyrosyl-373 radical pair (RP) formation primes α22 unfolding. Electron transfer-dependent protonation of aspartate-321 by tyrosine-373 is the epicenter of unfolding by disrupting salt bridges between α22 and the photolyase homology region. Before helix unfolding, another pathway opens transiently for FAD^•- protonation and RP stabilization. This link between RP formation and conformational changes provides a structural basis for signaling by animal-like cryptochromes.