Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein
Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein
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DOI：
10.1042/bcj20180803
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发表时间：
2019-01-15
影响因子：
4.1
通讯作者：
Versees, Wim
中科院分区：
文献类型：
--
作者：
Deyaert, Egon;Leemans, Margaux;Versees, Wim
LRR (leucine-rich repeat)-Roc（复杂蛋白的Ras）-COR （Roc的c末端）结构域是几乎所有Roco蛋白的核心作用，包括帕金森病相关蛋白LRRK2 （leucine-rich repeat kinase 2）。我们之前已经证明，在GTPase反应中，来自tepidum的Roco蛋白（CtRoco）经历了一个二聚体循环，蛋白质在无核苷酸和GDP（鸟苷-50-二磷酸）结合状态下主要是二聚体，而在GTP（鸟苷-50-三磷酸）结合状态下主要是单体。在这里，我们报道了CtRoco在无核苷酸状态下的晶体结构，首次显示了LRR-Roc-COR的排列。该结构揭示了紧凑的二聚体排列，并显示了二聚体界面中Roc GTPase结构域之间意想不到的密切相互作用，涉及p环，开关II环，G4区域和我们命名为“Roc二聚化环”的环的残基。氢-氘交换耦合质谱（HDX-MS）随后用于突出GTPase循环中单个步骤引起的结构变化。结构和HDX-MS数据提出了一条将核苷酸结合与单体化联系起来的途径，并通过Roc开关II将构象变化传递到LRR和COR结构域。总之，这项工作为Roco蛋白的调控提供了重要的新见解。
The LRR (leucine-rich repeat)-Roc (Ras of complex proteins)-COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimermonomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-50-diphosphate)-bound states and monomeric in the GTP (guanosine-50-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR-Roc-COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the 'Roc dimerization loop'. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.