Einfluss von Nukleotiden auf die biologische Aktivität von Cryptochrom 1 aus Arabidopsis thaliana

Cryptochromes (cry) are blue light receptors that together with photolyases form the so- called cryptochrome/photolyase family (CPF) with members occuring in all three domains of life. All members of the CPF show high structural similarities within the N-terminal photosensory (PHR, photolyase homologous region) domain that non-covalently binds the FAD cofactor. But their biologigal functions are different. While photolyases are enzymes that repair UV-damaged DNA in a light dependent manner, Cryptochromes lost the ability to repair DNA but gained important functions as photoreceptors to regulate a wide variety of biological responses. The two classical cryptochromes, cry1 and cry2, that are encoded in the model plant Arabidopsis thaliana have their main functions as blue-light photoreceptors that regulate photomorphogenesis and photoperiodic flowering. For the formation of the cryptochrome´s active state the photoexcitation of the flavin cofactor and a subsequent electron transfer towards the excited flavin is required. This allows the formation of the photoreceptors active state that, in case of plant cryptochromes, contains the flavin cofactor in its semireduced radical form FADH°. The transition from the resting state of the flavin in darkness to the active state is called „photoreduction“. For cry1 it was demonstrated for the first time in 2003 that plant cryptochromes bind ATP (Bouly et al., 2003). One year later the crystal structure of the PHR domain of Arabidopsis thaliana was published. Crystals were soaked with the non-hydrolyzable ATP analogon AMP-PNP and the co-crystal structure showed that the nucleotide is associated within the FAD cavity by a central tyrosine (Brautigam et al., 2004). Since then, not much is known about the nucleotide binding properties of cryptochromes. In 2010 a mutant of cry1 with a hyperactivity regarding blu-light responses was described (Exner et al., 2010). The mutation was caused by a leucine to phenylalanin exchange at position 407 of the cry1 PHR and was therefore called cry1L407F mutant. This mutant showed a cop-phenotype in the dark and based on in-silico structure modeling it was hypothesized that this mutant might mimic the signal active state of cry1 in darkness and that ATP binding might also be involved in this hyperactivity. The central aspect of this work was to investigate the effects of the binding of ATP and other nucleotides to the PHR domain of cry1 wild type and cry1L407F mutant protein. To have a suitable negative control in the performed assays, an ATP non-binding cry1 mutant was generated by using targeted mutagenesis. In this mutant, called cry1Y402A, the tyrosin on position 402, which is essential for ATP-binding in cry1, was exchanged to an alanin. The obtained data demonstrated that ATP enhances the photoreduction of both cry1WT and cry1L407F by stabilizing the active state of the photoreceptors. In protein stability assays it was shown that ATP binding also increases the stability of cry1. The data further indicated that the hyperactivity of the cry1L407F mutant is most likely caused by a structural alteration that is diffrent from the structure of cry1 with ATP bound. Furthermore a luciferase-based ATP-binding assay was established to approach the KD of ATP and cry1. This provided a basis for later-stage ITC meassurements in which a KD of 3.3 µM was determined for ATP binding to cry1 . Although many of the aspects of ATP binding of cryptochromes could be addressed the biological function of ATP binding remains unclear.