CRAF activation requires binding to membrane-anchored and active GTP-bound RAS. Whereas its RAS-binding domain (RBD) contains the main binding interface to the RAS G domain, its cysteine-rich domain (CRD) is responsible for association to anionic lipid-rich membranes. Both RAF domains are connected by a short linker, and it remains unclear if the two domains act independently or if one domain can impact the function of the other. Here, we used a combination of coarse-grained and all-atom molecular dynamics simulations of a CRAF RBD-CRD construct to investigate the dynamics of the RBD when it is tethered to CRD that is anchored to a POPC:POPS model membrane. First, we show that the RBD positioning is very dynamic with a preferential localization near the membrane surface. Next, we show that membrane-localized RBD has its RAS-binding interface mostly inaccessible because of its proximity to the membrane. Several positively charged residues in this interface were identified from simulations as important for driving RBD association to the membrane. Surface plasmon resonance (SPR) measurements confirmed that mutations of these RBD residues reduced the liposome partitioning of RBD-CRD. Last, simulations indicated that the presence of RBD near the membrane led to a local enrichment of anionic lipids that could potentially enhance the membrane affinity of the entire RBD-CRD construct. This was supported by SPR measurements that showed stronger liposome partitioning of RBD-CRD relative to CRD alone. These findings thus suggest that the RBD and CRD have synergistic effects on their membrane dynamics, with CRD bringing RBD closer to the membrane that impacts its accessibility to RAS and with RBD causing local anionic lipid enrichment that enhances the overall affinity between the membrane and RBD-CRD. These mechanisms have potential implications on the order of events of the interactions between RAS and CRAF at the membrane.

中文翻译：

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阴离子脂质影响 CRAF RBD-CRD RAS 结合位点可及性和膜结合亲和力


CRAF 激活需要与膜锚定且活性 GTP 结合的 RAS 结合。虽然其 RAS 结合结构域 (RBD) 包含与 RAS G 结构域的主要结合界面，但其富含半胱氨酸结构域 (CRD) 负责与阴离子富含脂质的膜结合。两个 RAF 结构域均通过短接头连接，目前尚不清楚这两个结构域是否独立发挥作用，或者一个结构域是否会影响另一个结构域的功能。在这里，我们结合使用 CRAF RBD-CRD 结构的粗粒度和全原子分子动力学模拟来研究当 RBD 与锚定在 POPC:POPS 模型膜上的 CRD 相连时 RBD 的动力学。首先，我们表明 RBD 定位是非常动态的，优先定位在膜表面附近。接下来，我们发现膜定位的 RBD 的 RAS 结合界面由于靠近膜而几乎无法接近。从模拟中发现，该界面中的几个带正电的残基对于驱动 RBD 与膜的结合非常重要。表面等离子共振 (SPR) 测量证实这些 RBD 残基的突变减少了 RBD-CRD 的脂质体分配。最后，模拟表明，膜附近 RBD 的存在导致阴离子脂质局部富集，这可能会增强整个 RBD-CRD 结构的膜亲和力。 SPR 测量结果支持了这一点，该测量显示 RBD-CRD 相对于单独的 CRD 具有更强的脂质体分配。 因此，这些发现表明，RBD 和 CRD 对它们的膜动力学具有协同作用，CRD 使 RBD 更接近膜，从而影响其与 RAS 的可及性，而 RBD 导致局部阴离子脂质富集，从而增强膜与 RBD 之间的整体亲和力。 CRD。这些机制对膜上 RAS 和 CRAF 之间相互作用的事件顺序具有潜在影响。