Einfluss von ATP auf die biologische Aktivität von Cryptochrom 2 aus Arabidopsis thaliana

Cryptochromes (cry) are blue light receptors that together with photolyases form the so-called cryptochrome/photolyase family with members occuring in all three domains of life. All family members show high structural similarities within the N-terminal photosensory (PHR, photolyase homologous region) domain that non-covalently bind the FAD cofactor, but show differences regarding their biological functions. Photolyases are DNA repair enzymes that remove UV-B lesions in damaged DNA. Cryptochromes lack DNA repair activity but gained important roles in regulating manifold biological responses. The model plant Arabidopsis thaliana encodes two classical cryptochromes, cry1 and cry2, with main functions in regulating photomorphogenesis and photoperiodic flowering. Prerequisite for active crys is electron transfer towards the excited flavin cofactor allowing the formation of the protein’s signalling state. In the case of plant crys the signalling state contains the semireduced radical form of the flavin, FADH°. The reduction of the resting state in the dark to the flavin’s lit state is called “photoreduction”. For cry1, Bouly et al. (Bouly et al., 2003) demonstrated for the first time that plant cryptochromes bind ATP. Subsequently, cry1’s metabolite binding site was determined in crystals of the PHR domain soaked with the non-hydrolyzable ATP analog AMP-PNP (Brautigam et al., 2004): The nucleotide is associated within the FAD cavity by a central tyrosine. However, still not much is known about the biochemical effects of ATP-binding to crys and consequences on their biological activity. The central topic of this work is the role of ATP binding to cry2. The residues in cry2 necessary for ATP-binding have not been ultimately identified by mutant studies. This is adressed in this PhD thesis by analyzing cry2 mutants with a replacement within the predicited ATP-binding site. Moreover, this work adresses, which effects ATP-binding has on cry2 in vitro. In addition, it was investigated how ATP-binding may affect the biological function of cry2 in planta. The recent results confirm that the among plant cryptochromes conserved Tyr residue 399 is essential in cry2 for ATP binding. By using in vitro assays with non-binding mutants, it could also been shown that ATP binding induces structural changes, especially in the C-terminal helix that connects the PHR domain with the C-terminal extension of the protein. Moreover, photoreduction in the cry2 wild-type protein is enhanced and FADH° is stabilized, when ATP is present. Complementation studies with transgenic Arabidopsis lines expressing the non-binding cry2 mutants in a cry2-deficient background showed that these proteins still retained biological activity. However, this remaining biological activity is reduced compaired to lines that express the wild-type protein. Consequently, ATP binding is not essential for cry2 but boosts the activity of cry2 by stabilizing the protein’s signalling state likely through conformational changes.