Hyperactivity of the Arabidopsis cryptochrome (cry1) L407F mutant is caused by a structural alteration close to the cry1 ATP-binding site

Abstract

Plant cryptochromes (cry) act as UV-A/blue light receptors. The prototype, cry1, regulates several light responses during the life cycle, including de-etiolation, and is also involved in regulating flowering time. The cry1 photocycle is initiated by light absorption by its FAD chromophore, which is most likely fully oxidized (FAD) in the dark state and photoreduced to the neutral flavin semiquinone (FADH°) in its lit state. Cryptochromes lack the DNA-repair activity of the closely related DNA photolyases, but they retain the ability to bind nucleotides such as ATP. The previously characterized L407F mutant allele of cry1 is biologically hyperactive and seems to mimic the ATP-bound state of cry1, but the reason for this phenotypic change is unclear. Here, we show thatL407F can still bind ATP, has less pronounced photoreduction and formation of FADH° than wild-type cry1, and has a dark reversion rate 1.7 times lower than that of the wild type. The hyperactivity of L407F is not related to a higher FADH° occupancy of the photoreceptor but is caused by a structural alteration close to the ATP-binding site. Moreover, we show that ATP binds to cry1 in both the dark and the lit states. This binding was not affected by cry1's C-terminal extension, which is important for signal transduction. Finally, we show that a recently discovered chemical inhibitor of cry1, 3-bromo-7-nitroindazole, competes for ATP binding and thereby diminishes FADH° formation, which demonstrates that both processes are important for cry1 function.

ATP boosts lit state formation and activity of Arabidopsis cryptochrome 2

“…Our previous studies on cry1 (Orth et al ., ) and cry2 (Engelhard et al ., ) have shown that ATP binding causes a strong acceleration of the formation of the flavin neutral semiquinone in these photoreceptors. This is concluded from the decrease in absorbance at 450 nm (contribution of FAD ox ), concomitant with an increase in absorbance at 520 nm (lit state; where only FADH o absorbs to a significant extent).…”

“…() applied ATP‐agarose chromatography. Based on the fact that the K D value obtained here was very close to that published by Ozgür and Sancar (), we consider our data relevant and also consistent with results obtained with cry1 Y402A (Orth et al ., ).…”

“…Besides ATP, we observed binding of other adenines to cry2, for example ADP and NAD (Figure S1), as shown previously for cry2 (Engelhard et al ., ) and cry1 (Orth et al ., ). Thus, it is not unlikely that different nucleotides bind to cry2 in vivo .…”

“…Thus, it is not unlikely that different nucleotides bind to cry2 in vivo . However, it was demonstrated for cry1 that adenines are the preferred ligands (Orth et al ., ). Since the ATP‐binding sites of cry1 and cry2 are highly conserved we propose that adenines are also the preferred ligands of cry2.…”

“…Taking the above‐mentioned effects of ATP on the photocycle and structure of cryptochrome into consideration, it appears likely that ATP binding is not only required for the demonstrated autophosphorylation activity of cry1 (Bouly et al ., ; Shalitin et al ., ; Ozgür and Sancar, ) but also for the primary activation mechanism of plant cryptochromes to boost light sensitivity. This notion is supported by conservation of residues involved in ATP binding of Arabidopsis cry1 in all cry proteins of the green lineage (Orth et al ., ).…”