STRUCTURAL STUDY OF ARF GTPASE REGULATORS AND EFFECTORS

ARF家族中的GTP酶调节囊泡运输事件，其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。 本研究的重点是通过X射线晶体学方法对ARF 1 GTP酶的三个配体（正调节因子SEC 7结构域、负调节因子ARFGAP和效应蛋白Beta-COP）进行结构表征;此外，还将通过对人ARF 1 GTP类似物和GDP结合形式的比较分析，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GTdR起作用的观点一致，初步工作已经证明Sec 7结构域和无核苷酸的ARF 1核心之间存在稳定的复合物。已获得的配合物晶体的分辨率至少为2.0 A，其结构将通过MR和MIR方法的组合来确定。 将这种结构与RasGAP的结构进行比较，这是在氨基酸序列中不相关的，将确定使GAP能够加速GTdR反应的共同特征。 类似物GppNHp。已经获得的晶体具有非常高的分辨率（至少1.4 A），并且通过分子置换解决了结构;晶体学改进正在进行中。 通过比较GppNHp和GDP结合的ARF的结构，可以进一步阐明ARF构象转换导致与效应分子发生GTP依赖性相互作用的机制。 总的来说，这些研究将提供深入了解通过ARF，ARFGAP和Sec 7结构域的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.