Mazzotta Gabriella Margherita, Bellanda Massimo, Minervini Giovanni, Damulewicz Milena, Cusumano Paola, Aufiero Simona, Stefani Monica, Zambelli Barbara, Mammi Stefano, Costa Rodolfo, Tosatto Silvio C E
Department of Biology, University of Padova, Padova, Italy.
Department of Chemical Sciences, University of Padova, Padova, Italy.
Front Mol Neurosci. 2018 Aug 20;11:280. doi: 10.3389/fnmol.2018.00280. eCollection 2018.
Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. We have previously shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. Here, we predict and demonstrate a novel Ca-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses we were able to identify and characterize a CaM binding motif in the dCRY C-terminus. Similarly, we also detailed the CaM binding site of the scaffold protein INAD and demonstrated that CaM bridges dCRY and INAD to form a ternary complex . Our results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM.
光是使大多数陆生生物体内源性计时系统同步的主要环境刺激因素。隐花色素（dCRY）是一种光响应黄素蛋白，可检测黎明和黄昏前后光强度和波长的变化。我们之前已经表明，dCRY以光依赖的方式通过无后电位失活D（INAD）作用于信号复合体，该复合体是一种包含视觉信号分子的多蛋白复合体，这表明dCRY在果蝇视觉中发挥作用。在此，我们预测并证明了dCRY与钙调蛋白（CaM）之间存在一种新的钙依赖性相互作用。通过酵母双杂交、免疫共沉淀（Co-IP）、核磁共振（NMR）和量热分析，我们能够鉴定并表征dCRY C末端的一个CaM结合基序。同样，我们还详细研究了支架蛋白INAD的CaM结合位点，并证明CaM连接dCRY和INAD形成三元复合体。我们的结果表明了一个过程，即快速的dCRY光反应刺激与INAD的相互作用，这可以通过一种由CaM调节的新机制进一步巩固。
