Structural Study of ARF-GTPase Regulators and Effectors

描述（由申请人提供）：ARF gtpase是囊泡运输步骤的关键调节剂，因此控制真核细胞的广泛过程，包括细胞内区室的维持和蛋白质和神经递质的胞外分泌。本提案的研究重点是对囊泡外壳组装和调节拆卸过程中ARF功能的综合理解。囊泡组装是由ARF对gtp依赖的囊泡外壳复合物的招募触发的。我们将通过确定在核苷酸交换反应中长期追求的中间体的晶体结构来探索ARF1上gdp到gtp转化的机制：ARF1- gdp与其Sec7催化剂络合，使用真菌毒素Brefeldin捕获：高质量晶体（2.4埃分辨率）。我们将通过分析arf -被膜相互作用来探索被膜募集的过程，包括对七聚体（550 kDa） COPI和四聚体（400 kDa） COPII被膜进行生化解剖。对于COPI，我们已经建立了六个亚复合物的蛋白表达系统，现在计划确定与“活性”COPI亚复合物结合的ARF1的结构。对于COPII外壳，我们之前确定了与ARF同源物Sar1结合的外壳内壳的晶体结构。我们现在已经解剖了COPII外壳生化，并将确定核心外壳复合物的晶体结构：高质量晶体（2.6埃分辨率）在手。最后，我们将通过关注触发这一过程的GTP水解反应来探索囊泡涂层的分解。我们之前发现，COPI可以将ARF1-ARFGAP复合物中GTP的水解加速300倍或更多。我们将通过在基态和过渡态配合物中结合ARF1-ARFGAP的COPI核的生化和晶体学分析来研究这种效应的基础。在COPII体系中，GTPase的最大速率加速需要内壳和外壳的催化作用。我们最近定义了一种能够最大速率加速的组装，包括内（Sec23）和外（Sec31片段）壳分子与Sar1 GTPase络合。我们已经在内部获得了这种复合物的小晶体，其衍射分辨率至少为2.8埃，我们正在努力改进。这些研究将为GTP依赖的水泡包被复合物组装和包被控制的GTP水解引发拆卸的过程提供见解。

DESCRIPTION (provided by applicant): ARF GTPases are key regulators of vesicle transport steps and thereby control a broad range of processes in eukaryotic cells, including the maintenance of intracellular compartments and the exocytosis of proteins and neurotransmitters. Research in this proposal focuses on an integrated understanding of ARF function in the processes of vesicular coat assembly and regulated disassembly. Vesicle assembly is triggered by the GTP-dependent recruitment of vesicular coat complexes by ARF. We will explore the mechanism of GDP-to-GTP conversion on ARF1 by determining the crystal structure of a long-sought-after intermediate in the nucleotide exchange reaction: ARF1-GDP complexed with its Sec7 catalyst, trapped using the fungal toxin, Brefeldin: high-quality crystals (2.4 Angstroms resolution) are in hand. We will explore the process of coat recruitment through analyses of ARF-coat interactions, involving the biochemical dissection of heptameric (550 kDa) COPI and tetrameric (400 kDa) COPII coats. For COPI, we have established protein expression systems for six subcomplexes, and plan now to determine the structure of ARF1 bound to an "active" COPI subcomplex. For the COPII coat, we previously determined the crystal structure of the inner shell of the coat bound to the ARF homolog, Sar1. We have now dissected the COPII outer shell biochemically, and will determine the crystal structure of a core outer-shell complex: high-quality crystals (2.6 Angstroms resolution) are in hand. Finally, we will explore vesicular coat disassembly by focussing on the GTP hydrolysis reaction that triggers this process. We previously discovered that COPI accelerates GTP hydrolysis in the ARF1-ARFGAP complex by 300-fold or more. We will investigate the basis for this effect by biochemical and crystallographic analysis of COPI core bound to ARF1-ARFGAP in ground- and transition-state complexes. In the COPII system, maximal rate acceleration of the GTPase requires catalytic contributions from both the inner and outer shells of the coat. We recently defined an assembly capable of maximal rate acceleration that includes inner (Sec23) and outer (Sec31 fragment) shell molecules complexed with Sar1 GTPase. We have obtained small crystals of this complex that diffract to at least 2.8 Angstroms resolution in-house, which we are working to improve. These studies will provide insight to the processes of GTP-dependent assembly of vesicular coat complexes and coat-controlled GTP hydrolysis triggering disassembly.