Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC, Canada.
Department of Pathology and Cell Biology, Faculty of Medicine, Université de Montréal, Montreal, QC, Canada.
Methods Mol Biol. 2024;2797:195-209. doi: 10.1007/978-1-0716-3822-4_14.
Knowledge of how effectors interact with RAS GTPases is key to understanding how these switch-like proteins function in cells. Effectors bind specifically to GTP-loaded RAS using RAS association (RA) or RAS binding domains (RBDs) that show wide-ranging affinities and thermodynamic characteristics. Both normal development and RAS-induced tumorigenesis depend on multiple distinct effector proteins that are frequently co-expressed and co-localized, suggesting an antagonistic nature to signaling whereby multiple proteins compete for a limited pool of activated GTPase. NMR spectroscopy offers a powerful approach to multiplex effectors and/or regulatory enzymes and quantifies their interaction with RAS, expanding our biophysical and systems-level understanding of RAS signaling in a more integrated and physiologically relevant setting. Here we describe a method to directly quantitate GTPase binding to competing effectors, using wild-type KRAS complex with ARAF and PLCε1 as a model. Unlabeled RBD/RA domains are added simultaneously to isotopically labeled RAS, and peak intensities at chemical shifts characteristic of individually bound domains provide quantitation. Similar competition-based assays can be run with small molecule interactors, GEF/GAP domains, or regulatory enzymes that drive posttranslational modifications. Such efforts bring in vitro interaction experiments in line with more complex cellular environments.
了解效应器如何与 RAS GTPases 相互作用是理解这些类似开关的蛋白质在细胞中如何发挥作用的关键。效应器使用 RAS 缔合 (RA) 或 RAS 结合结构域 (RBD) 特异性结合到加载 GTP 的 RAS 上，这些结构域具有广泛的亲和力和热力学特性。正常发育和 RAS 诱导的肿瘤发生都依赖于多种不同的效应蛋白，这些蛋白经常共表达和共定位，这表明信号传导具有拮抗性质，即多种蛋白竞争有限的激活 GTPase池。NMR 光谱提供了一种研究多功能效应器和/或调节酶的强大方法，并定量它们与 RAS 的相互作用，从而在更集成和生理相关的环境中扩展我们对 RAS 信号转导的生物物理和系统水平的理解。在这里，我们描述了一种直接定量竞争效应器与 GTPase 结合的方法，使用 ARAF 和 PLCε1 作为模型的野生型 KRAS 复合物。未标记的 RBD/RA 结构域同时添加到同位素标记的 RAS 中，并且化学位移特征为单独结合结构域的峰强度提供定量。可以使用小分子相互作用物、GEF/GAP 结构域或驱动翻译后修饰的调节酶运行类似的基于竞争的测定。这些努力使体外相互作用实验与更复杂的细胞环境保持一致。
