Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the distribution of dimers and higher-order oligomers of K-Ras4B, the most frequently mutated Ras isoform in human cancers. We focused on the constitutively active G12V K-Ras and two of its variants, K101E and K101C/E107C, which respectively destabilize and stabilize oligomers. Using raster image correlation spectroscopy and number and brightness analysis combined with fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and electron microscopy in live cells, we show that G12V K-Ras exists as a mixture of monomers, dimers and larger oligomers, while the K101E mutant is predominantly monomeric and K101C/E107C is dominated by oligomers. This observation demonstrates the ability of K-Ras to exist in multiple oligomeric states whose population can be altered by interfacial mutations. Using molecular modeling and simulations we further show that K-Ras uses two partially overlapping interfaces to form compositionally and topologically diverse oligomers. Our results thus provide the first detailed insight into the multiplicity, structure, and membrane organization of K-Ras homomers.

中文翻译：

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时空分析的K-Ras质膜相互作用揭示了多个高阶均聚物。

与质膜相关的Ras GTPases的自组装对细胞信号传导的调控具有重要意义。但是，均聚的结构基础和低聚状态的分数分布仍未确定。我们已经通过解释人类癌症中最常见的突变Ras亚型K-Ras4B的二聚体和高阶低聚物的分布来解决这些问题。我们专注于组成型活性G12V K-Ras及其两个变体K101E和K101C / E107C，它们分别使低聚物不稳定和稳定。使用光栅图像相关光谱法和数量和亮度分析，再结合活细胞中的漂白，荧光相关光谱法和电子显微镜后的荧光恢复，我们发现G12V K-Ras以单体混合物的形式存在，二聚体和更大的寡聚体，而K101E突变体主要是单体，而K101C / E107C则以寡聚体为主。该观察证明了K-Ras存在于多种低聚状态中的能力，这些低聚状态的种群可以通过界面突变来改变。使用分子建模和模拟，我们进一步表明K-Ras使用两个部分重叠的界面来形成组成和拓扑上不同的低聚物。因此，我们的结果提供了对K-Ras同聚物的多样性，结构和膜组织的首次详细了解。使用分子建模和模拟，我们进一步表明K-Ras使用两个部分重叠的界面来形成组成和拓扑上不同的低聚物。因此，我们的结果提供了对K-Ras同聚物的多样性，结构和膜组织的首次详细了解。使用分子建模和模拟，我们进一步表明K-Ras使用两个部分重叠的界面来形成组成和拓扑上不同的低聚物。因此，我们的结果提供了对K-Ras同聚物的多样性，结构和膜组织的首次详细了解。