Membrane Composition and Raf[CRD]-Membrane Attachment Are Driving Forces for K‑Ras4B Dimer Stability

Ras proteins are membrane-anchored GTPases that regulate key cellular signaling networks. It has been recently shown that different anionic lipid types can affect the properties of Ras in terms of dimerization/clustering on the cell membrane. To understand the effects of anionic lipids on key spatiotemporal properties of dimeric K-Ras4B, we perform all-atom molecular dynamics simulations of the dimer K-Ras4B in the presence and absence of Raf­[RBD/CRD] effectors on two model anionic lipid membranes: one containing 78% mol DOPC, 20% mol DOPS, and 2% mol PIP2 and another one with enhanced concentration of anionic lipids containing 50% mol DOPC, 40% mol DOPS, and 10% mol PIP2. Analysis of our results unveils the orientational space of dimeric K-Ras4B and shows that the stability of the dimer is enhanced on the membrane containing a high concentration of anionic lipids in the absence of Raf effectors. This enhanced stability is also observed in the presence of Raf­[RBD/CRD] effectors although it is not influenced by the concentration of anionic lipids in the membrane, but rather on the ability of Raf­[CRD] to anchor to the membrane. We generate dominant K-Ras4B conformations by Markov state modeling and yield the population of states according to the K-Ras4B orientation on the membrane. For the membrane containing anionic lipids, we observe correlations between the diffusion of K-Ras4B and PIP2 and anchoring of anionic lipids to the Raf­[CRD] domain. We conclude that the presence of effectors with the Raf­[CRD] domain anchoring on the membrane as well as the membrane composition both influence the conformational stability of the K-Ras4B dimer, enabling the preservation of crucial interface interactions.

Ras蛋白（Ras proteins）是一类锚定在膜上的GTP酶（GTPase），可调控关键的细胞信号转导网络。近期研究表明，不同类型的阴离子脂质会影响Ras蛋白在细胞膜上的二聚化与聚集特性。为阐明阴离子脂质对二聚体K-Ras4B关键时空特性的调控作用，本研究针对两种模式阴离子脂质膜体系，开展了有无Raf[RBD/CRD]效应蛋白（Raf[RBD/CRD]）存在下的二聚体K-Ras4B全原子分子动力学模拟。两种膜体系分别为：含78%摩尔分数二油酰磷脂酰胆碱（DOPC）、20%摩尔分数二油酰磷脂酰丝氨酸（DOPS）与2%摩尔分数磷脂酰肌醇二磷酸（PIP2）的膜，以及阴离子脂质浓度提升的膜（含50%摩尔分数DOPC、40%摩尔分数DOPS与10%摩尔分数PIP2）。对模拟结果的分析揭示了二聚体K-Ras4B的取向空间，并发现在无Raf效应蛋白的情况下，高阴离子脂质浓度的膜可增强二聚体的稳定性。在存在Raf[RBD/CRD]效应蛋白的体系中，同样观测到了这一稳定性提升现象；不过该稳定性并未受膜内阴离子脂质浓度的影响，而是取决于Raf[CRD]结构域锚定至膜上的能力。本研究通过马尔可夫态模型（Markov state modeling）构建了K-Ras4B的优势构象，并基于K-Ras4B在膜上的取向统计了各构象的占比。在含阴离子脂质的膜体系中，我们观测到K-Ras4B的扩散与PIP2的运动、以及阴离子脂质锚定至Raf[CRD]结构域之间存在相关性。本研究得出结论：具备膜锚定能力的Raf[CRD]结构域效应蛋白的存在，以及膜的组成成分，均可影响K-Ras4B二聚体的构象稳定性，从而维持其关键的界面相互作用。