Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome.
Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome.
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DOI：
10.1016/j.str.2022.03.010
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发表时间：
2022-06-02
期刊：
影响因子：
5.7
通讯作者：
Crane, Brian R.
中科院分区：
文献类型：
--
作者：
Lin, Changfan;Schneps, Connor M.;Chandrasekaran, Siddarth;Ganguly, Abir;Crane, Brian R.
隐花色素（CRY）通过与光中的永恒（TIM）结合来携带苍蝇的生物钟。响应于CRY黄素辅因子门TIM识别的光还原的螺旋C末端尾（CTT）的脱离。我们提出了一种普遍适用的选择无标记蛋白质相互作用（SWFTI）测定，允许定量CRY结合TIM在黑暗和光照。该测定被用来研究在黄素口袋中具有残基取代的CRY变体，并将它们的TIM亲和力与CTT解除对接相关联，如通过脉冲偶极ESR光谱法测量的和通过分子动力学模拟评估的。CRY变体与CTT去除或未对接的TIM组成型结合，而那些不能光还原结合TIM弱。响应于黄素氧化还原状态，两个保守的组氨酸残基通过介导CTT与黄素口袋和TIM的相互作用而促成了稳健的开/关开关。我们的方法提供了一种快速的手段来量化难以产生的蛋白质的相互作用。Lin等人提出了SWFTI方法，用于定量难以产生的蛋白质的相互作用。SWFTI用于表征生物钟光传感器隐花色素及其目标Timeless的光依赖性相互作用。SWFTI数据和生物物理测量表明，隐花色素黄素结合口袋undocks的自动抑制C-末端尾巴绑定永恒。
Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. We present a generally-applicable Select Western-blot-Free Tagged-protein Interaction (SWFTI) assay that allowed quantification of CRY binding to TIM in dark and light. The assay was utilized to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins. Lin et al. present the SWFTI method for quantifying interactions of difficult-to-produce proteins. SWFTI is used to characterize light-dependent interactions of the circadian clock photosensor Cryptochrome and its target, Timeless. SWFTI data and biophysical measurements reveal that the Cryptochrome flavin-binding pocket undocks an autoinhibitory C-terminal tail to bind Timeless.
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