Characterization of the Intrinsic and TSC2-GAP-Regulated GTPase Activity of Rheb by Real-Time NMR
Characterization of the Intrinsic and TSC2-GAP-Regulated GTPase Activity of Rheb by Real-Time NMR
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DOI：
10.1126/scisignal.2000029
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发表时间：
2009-01-27
影响因子：
7.3
通讯作者：
Stambolic, Vuk
中科院分区：
文献类型：
--
作者：
Marshall, Christopher B.;Ho, Jason;Stambolic, Vuk
结节性硬化症复合体2(TSC2)的基因在结节性硬化症中经常发生突变，它增加了小的异源三聚体GTP结合蛋白(G蛋白)Rheb的鸟苷三磷酸酶(GTPase)活性，从而导致细胞生长的主要调节因子雷帕霉素靶标(MTOR)的活性降低。在这里，我们描述了一种基于核磁共振(NMR)的定量实时检测方法的发展，以探索Rheb内在的和TSC2催化的GTPase活性的分子机制。我们证实，TSC2通过“天冬酰胺-拇指”机制替代无功能的Rheb的“催化”谷氨酰胺，使Rheb的GTP水解率提高了50倍，并且我们确定催化是由焓驱动的。我们研究的TSC2的大多数(但不是全部)疾病相关的GTP酶激活蛋白(GAP)结构域突变都会影响其酶活性。该方法可用于研究其他GTP酶的功能和调控。
Tuberous sclerosis complex 2 (TSC2), whose gene is frequently mutated in tuberous sclerosis, increases the guanosine triphosphatase (GTPase) activity of the small heterotrimeric GTP-binding protein (G protein) Rheb, thus resulting in the decreased activity of the mammalian target of rapamycin (mTOR), the master regulator of cell growth. Here, we describe the development of a nuclear magnetic resonance (NMR)-based, quantitative, real-time assay to explore the molecular mechanism of the intrinsic and TSC2-catalyzed GTPase activity of Rheb. We confirmed that TSC2 accelerated GTP hydrolysis by Rheb 50-fold through an "asparagine-thumb" mechanism to substitute for the nonfunctional "catalytic" glutamine of Rheb and we determined that catalysis was enthalpy driven. Most, but not all, of the disease-associated GTPase-activating protein (GAP) domain mutants of TSC2 that we examined affected its enzymatic activity. This method can now be applied to study the function and regulation of other GTPases.