Department of Molecular and Biomolecular Physics, National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donat Street, 400293 Cluj-Napoca, Romania.
Int J Mol Sci. 2024 Jan 24;25(3):0. doi: 10.3390/ijms25031430.
Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).
致癌 Ras 蛋白呈现出多种构象状态，这在大量的晶体结构报告中得到了证实。结合态的 GTP 被分为两种主要状态：“非活性”状态 1 和“活性”状态 2。最近关于 H-Ras 的报告表明，状态 2 表现出两种亚状态，这与 Tyr32 的取向直接相关：朝向 GTP 结合口袋和向外。在本文中，我们表明 N-Ras 表现出状态 2 的另一种亚状态，这与 Tyr32 的第三种取向有关，朝向 Ala18 并与 GTP 结合口袋平行。我们还表明，这种亚状态在 N-Ras 的 G12V 突变中高度采样，而在其野生型形式中几乎不存在，并且 G12V 突变禁止 GTP 酶激活蛋白 (GAP) 结合亚状态的采样，使这种突变具有致癌性。此外，使用分子动力学模拟，我们探索了膜对 N-Ras 构象状态动力学的重要性及其对 Ras 蛋白稳定性的强烈影响。此外，膜对 Ras 的构象（亚）状态采样有显著影响。这反过来又对 Ras 的激活/失活循环至关重要，因为鸟嘌呤核苷酸交换因子蛋白 (GEFs)/GTP 酶激活蛋白 (GAPs) 的结合。
