STRUCTURAL STUDY OF ARF GTPASE REGULATORS AND EFFECTORS

ARF家族中的GTP酶调控囊泡运输事件，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。这项建议的研究重点是通过X射线结晶学确定ARF1 GTPase的三个配体的结构特征：正调控域sec7结构域、负调控域ARFGAP和效应蛋白Beta-COP；此外，将通过比较分析人ARF1的GTP类似物和GDP结合形式来研究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GTPase的观点一致，初步工作证明了Sec7结构域和无核苷酸的ARF1核心之间存在稳定的复合体。该络合物的晶体的衍射率至少达到2.0A，其结构将由MR和MIR方法相结合确定。将该结构与在氨基酸序列中不相关的RasGAP的结构进行比较，将发现使GAP能够加速GTPase反应的共同特征。模拟的GppNHp。已经获得了衍射到非常高的分辨率(至少1.4A)的晶体，并通过分子置换解决了结构问题；结晶学的改进正在进行中。比较GppNHp和GDP结合的ARF的结构将建立ARF构象交换导致GTP依赖的与效应分子相互作用的机制。总体而言，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酶调控的一般结构原则。

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.