STRUCTURAL STUDY OF ARF GTPASE REGULATORS AND EFFECTORS

ARF家族中的gtpase调节囊泡转运事件，这些事件是广泛生理过程的基础，包括分泌和内吞作用以及胞内细胞器的生物发生。本课题主要研究了ARF1 GTPase的三种配体的x射线晶体学结构表征；正调控蛋白SEC7结构域、负调控蛋白ARFGAP和效应蛋白β - cop；此外，ARF构象转换的分子基础将通过对人类ARF1的gtp -模拟形式和gdp结合形式的比较分析来研究。与鸟嘌呤核苷酸交换因子（GEFs）通过稳定无核苷酸形式的GTPase起作用的观点一致，初步研究表明Sec7结构域和无核苷酸的ARF1核心之间存在稳定的复合物。该配合物的晶体衍射分辨率至少为2.0 A，其结构将通过MR和MIR方法的结合来确定。将该结构与氨基酸序列不相关的RasGAP结构进行比较，将发现使gap加速GTPase反应的共同特征。类似的，GppNHp。已经获得了衍射分辨率非常高（至少1.4 A）的晶体，并通过分子置换解决了结构；晶体学的细化正在进行中。通过比较GppNHp-和gdp结合的ARF的结构，可以建立ARF构象转换导致与效应分子gtp依赖相互作用的机制。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，以及支配ras相关GTPases调控的一般结构原理。

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.