A Comparative CEST NMR Study of Slow Conformational Dynamics of Small GTPases Complexed with GTP and GTP Analogues
A Comparative CEST NMR Study of Slow Conformational Dynamics of Small GTPases Complexed with GTP and GTP Analogues
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DOI：
10.1002/anie.201305434
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发表时间：
2013-10-04
影响因子：
16.6
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Long, Dong;Marshall, Christopher B.;Kay, Lewis E.
Ras超家族的小GTP酶是重要的细胞内信号分子，其功能由鸟苷核苷酸（GTP=三磷酸鸟苷和GDP=二磷酸鸟苷）的结合决定。[1]这些酶的GTP结合（“活性”）状态能够与特定的下游效应蛋白相互作用，从而引发广泛的细胞反应。[2，3]降低GTP水解速率并因此增加活性GTP结合状态的寿命的突变通常是致癌的，并有助于人类癌症的发展和转移。[4]优雅的31 P NMR研究GTP结合的Ras表明，酶之间的相互转换两个国家，一个次要的构象称为状态1和一个主要的物种指定状态2。[5-9]在其他Ras家族GTP酶中也观察到类似的构象动力学。[10-12]状态2通常被认为是能结合效应蛋白的构象，而状态1对这些分子表现出显著降低的亲和力。[5-7因此，低亲和力状态1的稳定被建议作为抑制Ras-效应物相互作用以减少致癌信号传导的策略。[15-17]例如，发现Zn 2 +-双（2-吡啶甲基）胺络合物[18]通过结合到变构位点来稳定状态1并抑制Ras-效应物相互作用，尽管具有低（毫摩尔）亲和力。Ras的内在GTP酶活性导致Ras· GTP在室温下几小时内转化为Ras· GDP，这对可以用生理GTP进行的实验的持续时间施加了实际限制。由于这个原因，稳定的GTP类似物，包括抗水解的鸟苷5-[B，g-亚氨基]三磷酸（GppNHp）、鸟苷5-O-[g-硫代]-三磷酸（GTPgS）和鸟苷5-[B，g-亚甲基]三磷酸（GppCH 2 p），已经用于活化的GTP酶的结构研究，包括Ras的状态1和2的表征以及它们相互转化的动力学和热力学。[5，6，8，14-19]然而，已知稳定GTP的这些修饰影响Ras的两种状态之间的构象平衡。[7-9]此外，通常用于探测核苷酸水解和交换速率的荧光团的附着可以显著地干扰几种GTP酶的这些过程的动力学。[20]因此，必须严格验证使用GTP类似物的小GTP酶研究的结论。一维31 P NMR光谱已被用于使用核苷酸作为探针探索许多GTP酶中的构象动力学[5-9]，特别关注GTP的g-磷，其对于状态1和2具有不同的化学位移。通过直接研究蛋白质来解决这些系统中的构象交换也是有意义的。O康纳和Kovrigin使用15 N弛豫色散[21，22]来表征室温下Ras· GppNHp中的毫秒主链波动。[23]然而，正如我们下面所描述的，同样的方法不能应用于生理Ras· GTP复合物。在这里，我们介绍了一种通用的方法，用于表征平衡构象交换的小GTP酶复合GTP使用交错的化学交换饱和转移（CEST）实验，有效地抑制系统误差所提取的交换参数引起的GTP水解。我们证明CEST光谱[24，25]是表征小GTP酶Ras和Rheb（脑中富集的Ras同系物）中缓慢构象交换的有力方法。动力学和…
The Ras superfamily of small GTPases are important intracellular signaling molecules, the functions of which are determined by the binding of guanosine nucleotides (GTP= guanosine triphosphate and GDP= guanosine diphosphate).[1] The GTP-bound (“active”) states of these enzymes are capable of interacting with specific downstream effector proteins, thus eliciting a wide range of cellular responses.[2, 3] Mutations that reduce the rate of GTP hydrolysis and thus increase the lifetime of the active GTP-bound state are frequently oncogenic and contribute to the development and metastasis of human cancers.[4] Elegant 31P NMR studies of GTP-bound Ras showed that the enzyme interconverts between two states, a minor conformer termed state 1 and a major species designated state 2.[5–9] Similar conformational dynamics have been observed in other Ras family GTPases as well.[10–12] State 2 is generally regarded as the conformation competent for binding effector proteins, whereas state1 exhibits significantly reduced affinity for these molecules.[5–7, 13, 14] Stabilization of the low-affinity state1 was hence suggested as a strategy to inhibit Ras–effector interactions so as to reduce oncogenic signaling.[15–17] For example, Zn2+–bis (2-picolyl) amine complexes [18] were found to stabilize state 1 and inhibit Ras–effector interactions by binding to an allosteric site, albeit with low (millimolar) affinity. The intrinsic GTPase activity of Ras leads to the conversion of Ras· GTP to Ras· GDP within a few hours at room temperature, imposing a practical limit on the duration of experiments that can be performed with physiological GTP. For this reason, stable GTP analogues, including guanosine 5-[b, g-imino] triphosphate (GppNHp), guanosine 5-O-[g-thio]-triphosphate (GTPgS), and guanosine 5-[b, g-methylene] triphosphate (GppCH2p) that resist hydrolysis have been used for structural studies of activated GTPases, including characterization of states1 and 2 of Ras and the kinetics and thermodynamics of their interconversion.[5, 6, 8, 14–19] However, these modifications that stabilize GTP are known to affect the conformational equilibrium between the two states of Ras.[7–9] Moreover, the attachment of the mant fluorophore commonly used to probe nucleotide hydrolysis and exchange rates can significantly perturb the kinetics of these processes for several GTPases.[20] Therefore, conclusions from studies of small GTPases using GTP analogues must be validated rigorously. One-dimensional 31P NMR spectroscopy has been used to explore conformational dynamics in a number of GTPases using the nucleotide as a probe,[5–9] focusing in particular on the g-phosphorous of GTP, which has distinct chemical shifts for states 1 and 2. It is also of interest to address conformational exchange in these systems through direct studies of the proteins as well. O Connor and Kovrigin used 15N relaxation dispersion [21, 22] to characterize the millisecond backbone fluctuations in Ras· GppNHp at room temperature.[23] However, as we describe below the same approach cannot be applied to the physiological Ras· GTP complex. Herein, we introduce a general approach for characterizing the equilibrium conformational exchange of small GTPases complexed with GTP using an interleaved chemical exchange saturation transfer (CEST) experiment that effectively suppresses systematic errors in extracted exchange parameters caused by GTP hydrolysis. We demonstrate that CEST spectroscopy [24, 25] is a powerful method for characterizing slow conformational exchange in the small GTPases Ras and Rheb (Ras homolog enriched in brain). The kinetics and …