BRAF 与 14-3-3 和 KRAS4B 复合物在纳米盘上的重构和特征描述。

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein Science Pub Date : 2024-06-01 DOI:10.1002/pro.5016
Ningdi F Liu, Masahiro Enomoto, Christopher B Marshall, Mitsuhiko Ikura
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引用次数: 0

摘要

RAF激酶是RAS-MAPK信号通路的关键组成部分,RAS-MAPK信号通路驱动细胞生长,在癌症中经常被过度激活。上游信号激活小 GTP 酶 RAS,RAS 将 RAF 募集到细胞膜上,推动后者从自动抑制的单体构象转变为活性二聚体。尽管最近取得了进展,但 RAF 激活的机理细节仍不清楚,特别是 RAS 和膜在介导 RAF 与 14-3-3 构象重排以允许 RAF 激酶域二聚化方面的作用。在这里,我们在纳米盘双层上重组了二聚 BRAF、14-3-3 二聚体和两个 KRAS4B 的活性复合物,并验证了其组装是 GTP 依赖性的。生物层干涉仪(BLI)被用来比较单体和二聚体全长 BRAF:14-3-3 复合物与 KRAS4B 连接的纳米盘(RAS-ND)的结合亲和力,并研究膜脂组成和 KRAS4B 的空间密度对结合的影响。1,2-二油酰-sn-甘油-3-磷酸-L-丝氨酸(DOPS)和较高的 KRAS4B 密度在不同程度上增强了 BRAF:14-3 与 RAS-ND 的相互作用,这取决于 BRAF 的低聚物状态。我们利用重组系统剖析了 KRAS4B 和膜对单体和二聚体 BRAF:14-3-3 复合物激酶活性的影响,发现单靠 KRAS4B 或纳米盘不足以刺激活性,而 RAS-ND 则提高了两种状态的 BRAF 的活性。全长 BRAF 与 14-3-3 和 KRAS 在无细胞、确定的脂质双分子层上的重组组装提供了一个更全面的生物物理视角,以探究膜表面对这种多聚信号复合物的调控。
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Reconstitution and characterization of BRAF in complex with 14-3-3 and KRAS4B on nanodiscs.

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.

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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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