Structural and functional characterization of fast-cycling RhoF GTPase.
Structural and functional characterization of fast-cycling RhoF GTPase.
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快速循环 RhoF GTPase 的结构和功能表征。
DOI：
10.1016/j.bbrc.2019.04.018
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Honda R.
中科院分区：
文献类型：
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作者：
Sugawara R;Ueda H;Honda R.
Ras超家族GTPases是一种在GDP结合的非活性状态和GTP结合的活性状态之间循环的分子开关，控制着许多信号通路。新出现的证据表明，包括RhoF在内的几种Ras超家族GTP酶不遵循经典的GDP/GTP交换循环;由于它们的GDP/GTP交换快速活性，它们充当组成型活性GTP结合蛋白（称为“快速循环”GTP酶）。为了了解快速循环GTP酶的分子基础，我们产生了具有GTP酶活性的重组RhoF，并检查了其功能和结构。在开关I/II区的两个点突变（Q77 L和P45 S，对应于Rac 1的Q61 L和P29 S）显著降低RhoF的GTP酶活性，表明RhoF和其他RAS超家族GTP酶之间的保守的GTP水解机制。然而，与先前的证据相反，在我们使用荧光标记的GDP的实验中，RhoF代表了缓慢的GDP/GTP交换活动，其在一天到一周的时间尺度上非常缓慢地解离GDP。缓慢的GDP解离被Mg 2+螯合和典型的快速循环突变F44 L（对应于Rac 1的F28 L）和P45 S加速。NMR和动态光散射数据揭示了RhoF的多聚体结构，其可以根据GTP/GDP结合状态在不同构象之间切换。总之，我们的研究表明（1）RhoF与其他RAS超家族GTP酶共享保守的GTP水解机制，但（2）RhoF采用独特的多聚体结构。我们的研究还认为，（3）RhoF的快速循环GTP酶的新兴概念应该使用不依赖于荧光标记GDP的替代测定法进行验证（251字）。
Ras superfamily GTPases are molecular switches that cycle between GDP-bound inactive state and GTP-bound active state to control many signaling pathways. Emerging evidence suggests that several Ras superfamily GTPases, including RhoF, do not follow the classical GDP/GTP exchange cycle; they act as constitutively active GTP-bound proteins due to their fast activities of GDP/GTP exchange (termed as ‘fast-cycling’ GTPases). To understand the molecular basis of the fast-cycling GTPases, we generated a GTPase active recombinant RhoF and examined its function and structure. Two point mutations in the switch I/II regions (Q77L and P45S, corresponding to Q61L and P29S of Rac1) significantly reduced the GTPase activity of RhoF, suggesting a conserved mechanism of GTP hydrolysis between RhoF and other RAS superfamily GTPases. However, in contrary to the previous evidence, RhoF represented a slow GDP/GTP exchange activity that dissociates GDP very slowly on a day-to-week time scale, in our experiment using fluorescently labeled GDP. The slow GDP dissociation was accelerated by Mg2+chelation and canonical fast-cycling mutations, F44L (corresponding to F28L of Rac1) and P45S. NMR and dynamic light scattering data revealed a multimeric structure of RhoF that can switch between different conformations depending on the GTP/GDP-bound state. Overall, our study suggests that (1) RhoF shares a conserved mechanism of GTP hydrolysis with other RAS superfamily GTPases, but (2) RhoF adopts a unique multimeric structure. Our study also argues that (3) the emerging concept of the fast-cycling GTPases for RhoF should be validated using an alternative assay that does not rely on fluorescently labeled GDP (251 words).