Table_1_Calmodulin Enhances Cryptochrome Binding to INAD in Drosophila Photoreceptors.DOCX

Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila 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 Ca2+-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 in vivo. 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.

光是大多数陆生生物体内源性计时系统的主要环境同步刺激因子。果蝇隐花色素（Drosophila cryptochrome，dCRY）是一种光响应黄素蛋白，可感知黎明与黄昏前后光强与波长的变化。我们此前的研究表明，dCRY可通过无后电位D失活蛋白（Inactivation No Afterpotential D，INAD）以光依赖方式作用于信号复合体（Signalplex）——这是一类包含视觉信号分子的多蛋白复合物，该结果提示dCRY在果蝇视觉中发挥功能。本研究中，我们预测并证实了dCRY与钙调蛋白（calmodulin，CaM）之间存在一种新型的钙离子依赖型相互作用。通过酵母双杂交、免疫共沉淀（Co-IP）、核磁共振（NMR）以及量热分析等实验手段，我们成功鉴定并表征了dCRY C端的一个钙调蛋白结合基序。同样，我们还明确了支架蛋白INAD的钙调蛋白结合位点，并证实钙调蛋白可介导dCRY与INAD形成体内（in vivo）三元复合物。我们的研究结果提示了这样一个过程：dCRY的快速光响应会触发其与INAD的相互作用，而这一相互作用可通过一种受钙调蛋白调控的新型机制得到进一步强化。