STRUCTURAL STUDY OF ARF GTPASE REGULATORS AND EFFECTORS

ARF GTPase 调节器和效应器的结构研究

• 批准号: 6386440
• 负责人: JONATHAN D GOLDBERG
• 金额: $ 23.34万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386440
• 关键词: X ray crystallography; biological signal transduction; conformation; crystallization; enzyme activity; enzyme mechanism; enzyme structure; guanine nucleotide binding protein; guanine nucleotide exchange factors; guanosine triphosphate; guanosinetriphosphatase activating protein; guanosinetriphosphatases; nucleotide analog; protein structure function; structural biology

GTPases in the ARF family regulate vesicle transport events that underlie a broad range physiological processes, including secretion and endocytosis and the biogenesis of intracellular organelles. Research in this proposal focuses on structural characteriztion, by X-ray crystallography, of threee ligands for ARF1 GTPase; the positive regulator SEC7 domain, the negative regulator ARFGAP, and the effector protein Beta-COP; Additionally, the moleuclar basis for ARF conformational switching will be studiedby a comparative analysis of the GTP-analog- and GDP-bound forms of human ARF1. Consistent with the view that guanine-nucleotide exchange factors (GEFs) act by stabilizing a nucleotide-free form of the GTPase, preliminary work has demonstrated a stable complex between the Sec7 domain and nucleotide-free ARF1 core. Crystals of the complex have been obatined that diffract to at least 2.0 A resolution the structure will be determined by a combination of MR and MIR methods. Comparison of this structure with that of RasGAP, which is unrealated in amino-acid sequence, will identify common features that enable GAPs to accelerate the GTPase reaction . The analog, GppNHp. Crystals have been obtained that diffrract to very high resolution ( at least 1.4 A), and the structure solved by molecular replacement; crystallographic refinement is in progress. Comparison of the structures of GppNHp- and GDP-bound ARF will exstablish the mechanism of ARF conformational swithching leads to GTP-dependent interactions with the effector molecule. Overall these studies will provide insight into both the specific mechanism of information transfer through ARF, ARFGAP and Sec7 domain, and into the gneral structural principles that govern the regulation of Ras-related GTPases.

ARF 家族中的 GTP 酶调节囊泡转运事件，这些事件是广泛的生理过程的基础，包括分泌和内吞作用以及细胞内细胞器的生物发生。 本提案的研究重点是通过 X 射线晶体学对 ARF1 GTPase 的三个配体进行结构表征；正调节因子 SEC7 结构域、负调节因子 ARFGAP 和效应蛋白 Beta-COP；此外，将通过人 ARF1 的 GTP 类似物和 GDP 结合形式的比较分析来研究 ARF 构象转换的分子基础。与鸟嘌呤核苷酸交换因子 (GEF) 通过稳定无核苷酸形式的 GTP 酶发挥作用的观点一致，初步工作已证明 Sec7 结构域和无核苷酸 ARF1 核心之间存在稳定的复合物。已获得复合物的晶体，其衍射分辨率至少为 2.0 A，结构将通过 MR 和 MIR 方法的组合来确定。 将此结构与氨基酸序列无关的 RasGAP 的结构进行比较，将确定使 GAP 能够加速 GTPase 反应的共同特征。 类似物，GppNHp。获得了衍射分辨率非常高（至少 1.4 A）的晶体，并且通过分子置换解析了结构；晶体学细化正在进行中。 比较 GppNHp 和 GDP 结合的 ARF 的结构将建立 ARF 构象转换导致与效应分子依赖 GTP 相互作用的机制。 总的来说，这些研究将深入了解通过 ARF、ARFGAP 和 Sec7 结构域进行信息传递的具体机制，以及控制 Ras 相关 GTP 酶调节的一般结构原理。