Unraveling the molecular circuits controlling plant cryptochrome activity

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The physiological responses controlled by the cryptochrome (cry) blue light photoreceptors in plants are well studied and several of the downstream signaling components were identified. However, understanding the primary molecular mechanisms of how the light-induced activity of cryptochromes is achieved is still in its infancy. More recently it became evident that plant cryptochromes bind nucleotides such as ATP. Binding of these metabolites boosts the accumulation of the cryptochrome signaling state, and stabilizes the protein by inducing structural changes. In line with an important role of nucleotide binding for stimulating cryptochrome activity is the observation that a cry1 mutant (cry1L407F), which mimics the ATP-bound state, is constitutively active. The opposite process, inactivation of cryptochrome, is likewise not fully understood. It is hypothesized that re-oxidation through molecular oxygen of the semireduced FAD chromophore is the initial event in inactivation of cryptochrome. However, very recent data showed that plant cryptochromes dimerize upon photoactivation, that only the dimeric state is active, and that dimerization is inhibited by proteins named Blue light Inhibitors of Cryptochromes (BICs). We propose a regulatory circuit for controlling cryptochrome activity involving nucleotide and BIC binding to cryptochromes. This model will be tested in vitro using recombinant and purified cry wild type, hyperactive cry1L407F and BIC proteins to see how light, ATP and BICs affect the dimerization of crys. A mechanistic understanding of BIC function is facilitated by our finding that these proteins bind iron. We will investigate structural changes in cry1 caused by ATP, light and BIC-binding using hydrogen-deuterium exchange mass spectrometry and eventually X-ray crystallography. Finally, we will validate our in vitro data in planta by expressing our available non ATP-binding mutants of cry1 and cry2 in Arabidopsis and analyze their biological activity.