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 inter-domain modulation would be critical for CRYs' ability to transduce a blue-light 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.

Fig. 1

(a) TG signal of the full-length AtCRY1 after blue-light excitation at 465 nm in the buffer solution containing 50 mM Tris, 500 mM NaCl, and 30% glycerol (pH 7.5) at q² = 4.1 × 10¹¹ m⁻² at the excitation wavelength of 465 nm (red line). The signal was measured at a concentration of 80 μM, at 12 °C, and at an excitation laser power of ~10 μJ. (inset) Magnification of the signal in the time region of 2 μs to 40 ms. (b). TG signals of full-length AtCRY1 at various q² values: 4.1 × 10¹¹ m⁻² (red), 1.9 × 10¹¹ m⁻² (orange), 7.3 × 10¹⁰ m⁻² (green), and 3.5 × 10¹⁰ m⁻² (blue). Signals were normalized by thermal grating intensity. (c) Magnification of (b) in the time region of 100 μs to 150 ms. The q²-independent decay components were observed in the millisecond time range (arrows). These components indicate reaction kinetics (absorption changes). The black broken curves in these figures show the best fitted curves by Eq. (4).

Fig. 2

The comparison of single-exponential (black continuous curve) and bi-exponential (black broken curve) analyses for the weak species grating components observed in the TG signal of full-length AtCRY1 at q² = 7.3 × 10¹⁰ m⁻². Residual errors are also shown in the figure top by dark-green continuous curve (singleexponential analysis) and green broken curve (biexponential analysis).

Fig. 3

(a) TG signals for the full-length AtCRY1 at protein concentrations of 80 μM (red), 64 μM (orange), 50 μM (green), and 39 μM (blue) at q² = 4.3 × 10¹¹ m⁻². The TG signals were normalized by the diffusion peak. (b) AtCRY1 absorption spectra at matching concentrations.

Fig. 4

TG signal of the wild-type full-length AtCry1 (black), the CCT domain truncated AtCry1 (blue), and W324F mutant (red) of full-length AtCry1 at q² = 2.3 × 10¹² m⁻² under the same experimental conditions. The signals were measured at protein concentrations of 50 μM, at 12 °C.

Fig. 5

Partial proteolysis of the full-length AtCRY1 under dark (left) and light (right) conditions. The bands showing differential proteolysis are indicated (arrows). The topmost band is less stable subsequent to illumination.

Fig. 6

The schematic illustrations of the proposed photoreaction of AtCRY1. In the dark, AtCRY1 takes a relatively compact structure by inter-domain interactions. Blue light induces the reduction of FAD chromophore to the radical form (FADH·) through the intra-protein electron transfer from the conserved tryptophan triad. The change in the PHR-like domain could propagate to the interface between the PHR-like and the CCT domains, resulting in their dissociation. This dissociation would increase the surface area (colored pink) between AtCRY1 and the solvent, producing a reduction in the molecular diffusion coefficient and the protein stability to the protease. This simultaneously would expose interaction sites for other molecules, thereby activating signals in the photomorphogenesis pathways.