Unraveling the molecular circuits controlling plant cryptochrome activity

植物中隐花色素（cry）蓝光感受器调控的生理反应得到了很好的研究，并鉴定了几种下游信号成分。然而，了解隐色素如何实现光诱导活性的主要分子机制仍处于起步阶段。最近发现，植物隐色素可以结合核苷酸，如ATP。这些代谢物的结合促进了隐色素信号状态的积累，并通过诱导结构变化来稳定蛋白质。与核苷酸结合对刺激隐花色素活性的重要作用相一致的是，观察到cry1突变体（cry1L407F）模仿atp结合状态，具有组成性活性。相反的过程，隐花色素的失活，也同样不完全清楚。据推测，通过半还原FAD发色团的分子氧再氧化是隐花色素失活的初始事件。然而，最近的数据表明，植物隐色素在光激活下二聚化，只有二聚体状态是活跃的，并且二聚化被一种名为Blue light Inhibitors of cryptochromes （bic）的蛋白质所抑制。我们提出了一个控制隐色素活性的调控回路，涉及核苷酸和BIC与隐色素的结合。该模型将在体外使用重组和纯化的cry野生型、高活性cry1L407F和BIC蛋白进行测试，以观察光、ATP和BIC如何影响cry的二聚化。我们发现这些蛋白质与铁结合，从而促进了对BIC功能的机制理解。我们将利用氢-氘交换质谱和x射线晶体学研究ATP、光和bic结合引起的cry1结构变化。最后，我们将通过在拟南芥中表达cry1和cry2的非atp结合突变体来验证我们的体外数据，并分析它们的生物活性。

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.