Allosteric modulation of the catalytic VYD loop in Slingshot by its N-terminal domain underlies both Slingshot auto-inhibition and activation
Allosteric modulation of the catalytic VYD loop in Slingshot by its N-terminal domain underlies both Slingshot auto-inhibition and activation
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Slingshot 中催化 VYD 环通过其 N 端结构域的变构调节是 Slingshot 自动抑制和激活的基础
DOI：
10.1074/jbc.ra118.004175
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发表时间：
2018-08
影响因子：
4.8
通讯作者：
Sun Jinpeng
中科院分区：
文献类型：
--
作者：
Yang Duxiao;Xiao Peng;Li Qing;Fu Xiaolei;Pan Chang;Lu Di;Wen Shishuai;Xia Wanying;He Dongfang;Li Hui;Fang Hao;Shen Yuemao;Xu Zhigang;Lin Amy;Wang Chuan;Yu Xiao;Wu Jiawei;Sun Jinpeng
弹弓是调节细胞骨架动力学的磷酸酶，其活性在不同的生理环境中受到严格的调节。最近，异常升高的弹弓活性已经涉及许多人类疾病，如癌症、阿尔茨海默病和血管疾病。因此，弹弓特异性抑制剂具有治疗潜力。然而，缺乏对弹弓的催化机制及其被肌动蛋白激活的酶学理解。在这里，我们报告说，N-末端区域的人弹弓2自动抑制其磷酸酶活性的非竞争性方式。pH依赖性磷酸酶测定和离去基团依赖性研究表明，N-末端结构域的弹弓2调节催化过程中的产物的离去基团的稳定性，通过调节催化VYD环中的一般酸Asp 361。F-肌动蛋白结合解除了这种自抑制，并恢复了一般酸的功能。有限的胰蛋白酶消化和生物物理学研究确定了F-肌动蛋白结合后弹弓2的大构象变化。包括Leu 63在内的N-末端结构元件的解离以及磷酸酶结构域的α-螺旋-2和β-折叠-3之间的环的暴露充当了通过体外F-肌动蛋白结合和通过细胞中神经调节蛋白刺激进行弹弓激活的结构基础。此外，我们还设计了一种基于FlAsH-BRET的Slingshot 2生物传感器，其读数与Slingshot 2的体内磷酸酶活性高度相关。我们的研究结果揭示了人类弹弓磷酸酶的自抑制机制和变构激活机制。他们还有助于设计新的策略来研究弹弓调节在各种细胞环境中，并筛选新的激活剂/抑制剂的弹弓活动。
Slingshots are phosphatases that modulate cytoskeleton dynamics, and their activities are tightly regulated in different physiological contexts. Recently, abnormally elevated Slingshot activity has been implicated in many human diseases, such as cancer, Alzheimer's disease, and vascular diseases. Therefore, Slingshot-specific inhibitors have therapeutic potential. However, an enzymological understanding of the catalytic mechanism of Slingshots and of their activation by actin is lacking. Here, we report that the N-terminal region of human Slingshot2 auto-inhibits its phosphatase activity in a noncompetitive manner. pH-dependent phosphatase assays and leaving-group dependence studies suggested that the N-terminal domain of Slingshot2 regulates the stability of the leaving group of the product during catalysis by modulating the general acid Asp361 in the catalytic VYD loop. F-actin binding relieved this auto-inhibition and restored the function of the general acid. Limited tryptic digestion and biophysical studies identified large conformational changes in Slingshot2 after the F-actin binding. The dissociation of N-terminal structural elements, including Leu63, and the exposure of the loop between α-helix-2 and β-sheet-3 of the phosphatase domain served as the structural basis for Slingshot activation via F-actin binding in vitro and via neuregulin stimulation in cells. Moreover, we designed a FlAsH-BRET–based Slingshot2 biosensor whose readout was highly correlated with the in vivo phosphatase activities of Slingshot2. Our results reveal the auto-inhibitory mechanism and allosteric activation mechanisms of a human Slingshot phosphatase. They also contribute to the design of new strategies to study Slingshot regulation in various cellular contexts and to screen for new activators/inhibitors of Slingshot activity.
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影响因子：
4.8
作者：
K. Xiao;S. Shenoy;K. Nobles;R. Lefkowitz
通讯作者：
K. Xiao;S. Shenoy;K. Nobles;R. Lefkowitz
DOI：
--
发表时间：
2015
期刊：
--
影响因子：
--
作者：
Qi Wang
通讯作者：
Qi Wang
抑制蛋白偏向的 AT1R 激动作用通过 TRPC3 偶联诱导急性儿茶酚胺分泌
影响因子：
16.6
作者：
Liu CH;Gong Z;Liang ZL;Liu ZX;Yang F;Sun YJ;Ma ML;Wang YJ;Ji CR;Wang YH;Wang MJ;Cui FA;Lin A;Zheng WS;He DF;Qu CX;Xiao P;Liu CY;Thomsen AR;Joseph Cahill T 3rd;Kahsai AW;Yi F;Xiao KH;Xue T;Zhou Z;Yu X;Sun JP
通讯作者：
Sun JP
影响因子：
2.9
作者：
Hwangseo Park;So Ya Park;S. Ryu
通讯作者：
Hwangseo Park;So Ya Park;S. Ryu
影响因子：
21.3
作者：
Eiseler T;Döppler H;Yan IK;Kitatani K;Mizuno K;Storz P
通讯作者：
Storz P