Light-Induced Conformational Changes in Full-Length Arabidopsis thaliana Cryptochrome

Abstract

Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This interdomain modulation would be critical for CRYs’ ability to transduce a bluelight signal into altered protein–protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.

Magnetically sensitive light-induced reactions in cryptochrome are consistent with its proposed role as a magnetoreceptor

“…If these processes were much faster than 1 μs there would be insufficient time for the geomagnetic field to influence the spin dynamics; if they were much slower, the effect would almost certainly be attenuated by spin-decoherence (14). It seems unlikely, however, that the conformational changes needed to generate the signaling state [probably rearrangement of the Cterminal domain (46)] could be as fast as 10 μs. Our results show that formation of a secondary species (RP2) from the magnetically sensitive radical pair RP1, [FAD •− TrpH •þ ], avoids the need for an abnormally rapid protein rearrangement or exceptionally slow spin-decoherence.…”

Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome

“…Type I Cryptochromes-The decisive steps of signal generation in the plant cryptochrome between the initial ultrafast electron transfer to FAD (14) and the structural change in the CCT (24,25) are unclear. In particular, the role of the proton transfer to the flavin radical needs to be elucidated considering the fact that the aspartic acid close to flavin is exclusively present in the plant cryptochrome.…”

“…Therefore, proton transfer stabilizes the radical state for the time range later than a few milliseconds even under aerobic conditions. This time range is crucial for the signaling process because of the unfolding of the CCT with a time constant of 400 ms (25). It is conceivable, in general, that such a conformational change occurs after relaxation of the initiating photochemical process in the cofactor.…”

“…Illumination of plant cryptochrome leads to conformational changes in the PHR (23) and in the CCT (24). The latter are associated with a significant change in the diffusion coefficient with a time constant of 400 ms (25).…”

Blue-light dependent ROS formation by Arabidopsis cryptochrome-2 may contribute toward its signaling role

“…In this way, we provide evidence that significant structural rearrangement of the Atcry2 C-terminus follows from light activation, similarly to what has been previously reported for Arabidopsis cry1. 9 Blue-light induced accumulation of ROS in response to light activation of cry2…”