Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.
BMC Biol. 2019 Oct 9;17(1):77. doi: 10.1186/s12915-019-0698-5.
Ryanodine receptor (RyR), a calcium-release channel located in the sarcoplasmic reticulum membrane of muscles, is the target of insecticides used against a wide range of agricultural pests. Mammalian RyRs have been shown to be under the regulatory control of several kinases and phosphatases, but little is known about the regulation of insect RyRs by phosphorylation.
Here we present the crystal structures of wild-type and phospho-mimetic RyR Repeat34 domain containing PKA phosphorylation sites from diamondback moth (DBM), a major lepidopteran pest of cruciferous vegetables. The structure has unique features, not seen in mammalian RyRs, including an additional α-helix near the phosphorylation loop. Using tandem mass spectrometry, we identify several PKA sites clustering in the phosphorylation loop and the newly identified α-helix. Bioinformatics analysis shows that this α-helix is only present in Lepidoptera, suggesting an insect-specific regulation. Interestingly, the specific phosphorylation pattern is temperature-dependent. The thermal stability of the DBM Repeat34 domain is significantly lower than that of the analogous domain in the three mammalian RyR isoforms, indicating a more dynamic domain structure that can be partially unfolded to facilitate the temperature-dependent phosphorylation. Docking the structure into the cryo-electron microscopy model of full-length RyR reveals that the interface between the Repeat34 and neighboring HD1 domain is more conserved than that of the phosphorylation loop region that might be involved in the interaction with SPRY3 domain. We also identify an insect-specific glycerol-binding pocket that could be potentially targeted by novel insecticides to fight the current resistance crisis.
The crystal structures of the DBM Repeat34 domain reveals insect-specific temperature-dependent phosphorylation sites that may regulate insect ryanodine receptor function. It also reveals insect-specific structural features and a potential ligand-binding site that could be targeted in an effort to develop green pesticides with high species-specificity.
Ryanodine 受体（RyR）是一种位于肌肉肌浆网膜上的钙释放通道，是用于防治多种农业害虫的杀虫剂的靶标。已经证明哺乳动物 RyR 受几种激酶和磷酸酶的调控，但关于磷酸化对昆虫 RyR 的调控知之甚少。
本文展示了来自小菜蛾（一种十字花科蔬菜的主要鳞翅目害虫）的野生型和磷酸模拟 RyR Repeat34 结构域，该结构域含有 PKA 磷酸化位点。该结构具有一些独特的特征，在哺乳动物 RyR 中没有发现，包括在磷酸化环附近的另一个α-螺旋。通过串联质谱分析，我们在磷酸化环和新发现的α-螺旋中鉴定了几个 PKA 位点。生物信息学分析表明，该α-螺旋仅存在于鳞翅目昆虫中，表明存在昆虫特异性调节。有趣的是，这种特定的磷酸化模式是温度依赖性的。与三种哺乳动物 RyR 同工型的类似结构域相比，DBM Repeat34 结构域的热稳定性明显较低，表明结构域具有更动态的结构，可以部分展开以促进温度依赖性磷酸化。将该结构对接进全长 RyR 的冷冻电镜模型中表明，Repeat34 和相邻 HD1 结构域之间的界面比参与与 SPRY3 结构域相互作用的磷酸化环区域更保守。我们还鉴定了一个昆虫特异性的甘油结合口袋，可能成为新型杀虫剂的潜在靶标，以应对当前的抗药性危机。
DBM Repeat34 结构域的晶体结构揭示了昆虫特异性的温度依赖性磷酸化位点，可能调节昆虫 Ryanodine 受体的功能。它还揭示了昆虫特异性的结构特征和潜在的配体结合口袋，这可能成为开发具有高物种特异性的绿色杀虫剂的目标。
