Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, United States.
NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland 21701, United States.
Biochemistry. 2024 Nov 5;63(21):2740-2749. doi: 10.1021/acs.biochem.4c00116. Epub 2024 Oct 9.
KRas4b is a small plasma membrane-bound G-protein that regulates signal transduction pathways. The interaction of KRas4b with the plasma membrane is governed by both its basic C-terminus, which is farnesylated and methylated, and the lipid composition of the membrane itself. The signaling activity of KRas4b is intricately related to its interaction with various binding partners at the plasma membrane, underlining the critical role played by the lipid environment. The calcium-binding protein calmodulin binds farnesylated KRas4b and plays an important role in the dynamic spatial cycle of KRas4b trafficking in the cell. We utilize Biolayer Interferometry to assay the role of lipid headgroup, chain length, and electrostatics in the dissociation kinetics of fully post-translationally modified KRas4b from Nanodisc bilayers with defined lipid compositions. Our results suggest that calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS containing bilayers. In addition to this headgroup dependence, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with a matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers, showing that the overall charge of the bilayer, the identity of the headgroups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.
KRas4b 是一种小型质膜结合 G 蛋白，可调节信号转导途径。KRas4b 与质膜的相互作用受其碱性 C 端的调节，该 C 端既被法呢基化又被甲基化，以及膜本身的脂质组成。KRas4b 的信号活性与它在质膜上与各种结合伴侣的相互作用密切相关，这突显了脂质环境所起的关键作用。钙结合蛋白钙调蛋白与法尼基化的 KRas4b 结合，并在 KRas4b 在细胞中的动态空间循环中发挥重要作用。我们利用生物层干涉技术来测定脂质头基、链长和静电作用在完全翻译后修饰的 KRas4b 从具有明确定义脂质组成的纳米盘双层中解离动力学中的作用。我们的结果表明，钙调蛋白促进 KRas4b 从阴离子膜上解离，与含有 PIP2 的双层相比，KRas4b 从 PS 中相对较慢地置换。除了这种头基依赖性外，与具有匹配酰链长度的脂质相比，KRas4b 从纳米盘中的脂质组成包含不匹配的不饱和酰链时，其解离似乎更慢。这些发现有助于理解脂质组成在 KRas4b 结合和从脂质双层释放中的作用，表明双层的总电荷、存在的头基的身份以及酰链的长度和饱和度在 KRas4b 从膜释放中起着关键作用，这可能为针对 Ras-膜相互作用的治疗干预提供了新的思路。
