Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix.
Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix.
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DOI：
10.1021/acschembio.0c00543
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发表时间：
2020-10-16
影响因子：
4
通讯作者：
Tonge PJ
中科院分区：
文献类型：
--
作者：
Iuliano JN;Collado JT;Gil AA;Ravindran PT;Lukacs A;Shin S;Woroniecka HA;Adamczyk K;Aramini JM;Edupuganti UR;Hall CR;Greetham GM;Sazanovich IV;Clark IP;Daryaee T;Toettcher JE;French JB;Gardner KH;Simmerling CL;Meech SR;Tonge PJ
光激活蛋白结构域为光遗传学和光生物学提供了一种方便的、模块化的、可遗传编码的传感器。虽然这些领域现在已经部署在许多系统中，光活化的精确机制和伴随的结构动力学，调节输出域的活动仍然有待充分阐明。在植物向光蛋白（LOV 2）的C-末端光、氧、电压（LOV）结构域中，蓝光激活导致保守的Cys残基与嵌入的FMN发色团之间形成加合物，保守的Gln（Q513）旋转，以及与输出结构域偶联的螺旋（Jα-helix）解折叠。本论文采用分子动力学模拟、时间分辨红外光谱、溶液核磁共振光谱和细胞内光遗传学实验等多学科方法，对燕麦LOV 2（AsLOV 2）中Jα螺旋解折叠的变构途径进行了研究。在暗态下，N414的侧链与Q513的主链N-H氢键合。模拟预测了在Cys加合物形成后Q513的类α运动，导致N414的侧链和Q513的主链C=O之间的相互作用丧失，并且在Q513和N414侧链之间形成瞬时氢键。通过定点诱变评价N414在信号转导中的中心作用，支持Jα螺旋解折叠动力学与Zdk 2-AsLOV 2光遗传构建体的细胞功能之间的直接联系。通过这种多方面的方法，我们表明Q513和N414是蛋白质结构动力学的关键介质，将FMN发色团的超快（亚ps）激发与导致光感受器激活和生物学功能的微秒构象变化联系起来。
Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.
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影响因子：
2.9
作者：
Iuliano JN;Gil AA;Laptenok SP;Hall CR;Tolentino Collado J;Lukacs A;Hag Ahmed SA;Abyad J;Daryaee T;Greetham GM;Sazanovich IV;Illarionov B;Bacher A;Fischer M;Towrie M;French JB;Meech SR;Tonge PJ
通讯作者：
Tonge PJ
光遗传学的蓝光受体。
影响因子：
62.1
作者：
Losi A;Gardner KH;Möglich A
通讯作者：
Möglich A
DOI：
10.1126/science.1159052
发表时间：
2008-10-17
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Lee J;Natarajan M;Nashine VC;Socolich M;Vo T;Russ WP;Benkovic SJ;Ranganathan R
通讯作者：
Ranganathan R
影响因子：
2.9
作者：
Harper, SM;Christie, JM;Gardner, KH
通讯作者：
Gardner, KH
影响因子：
16.6
作者：
Chang, Xue-Ping;Gao, Yuan-Jun;Thiel, Walter
通讯作者：
Thiel, Walter