Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas.
Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas; Biochemistry and Cell Biology Program, MD Anderson University of Texas Health Science Center at Houston, Graduate School of Biochemical Sciences, Houston, Texas.
Biophys J. 2019 Jan 22;116(2):179-183. doi: 10.1016/j.bpj.2018.12.011. Epub 2018 Dec 20.
Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal β-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 μs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.
最近的研究表明，小分子 GTPase KRAS 相对于阴离子模型膜的平面可以采取多种取向，其中催化结构域的三个 C 末端螺旋或三个 N 末端 β 链面向膜。这具有功能意义，因为在后一种情况下，膜会阻塞效应物相互作用的表面。然而，目前尚不清楚膜的重新取向是如何发生的，更关键的是，它是否会发生在作为信号通路中分子开关的 KRAS 发挥作用的细胞中。在此，我们使用来自癌基因 G12V-KRAS 突变体在磷脂酰胆碱/磷脂酰丝氨酸双层中进行的 20 μs 长原子分子动力学模拟的数据，首先表明 KRAS 中柔性区域的内部构象波动导致了三种不同的膜取向。然后，我们使用源自仓鼠肾细胞的天然脂质纳米盘的单分子荧光共振能量转移测量，表明 G12V-KRAS 样品对应于预测取向的三种构象状态。综合结果表明，相对较小的能量势垒分离取向状态，并且信号竞争构象占主导地位。
