Structural Study of ARF-GTPase Regulators and Effectors

描述（由申请人提供）：ARF GTP酶是囊泡转运步骤的关键调节剂，从而控制真核细胞中的广泛过程，包括细胞内区室的维持以及蛋白质和神经递质的胞吐作用。在这个建议的研究集中在一个综合的理解ARF功能的过程中泡状外套组装和调节拆卸。ARF通过GTP依赖性募集囊泡外壳复合物来触发囊泡组装。我们将通过确定核苷酸交换反应中长期寻求的中间体的晶体结构来探索ARF 1上GDP转化为GTP的机制：ARF 1-GDP与其Sec 7催化剂复合，使用真菌毒素Brefeldin捕获：高质量晶体（2.4埃分辨率）在手。我们将通过分析ARF-外套相互作用，包括七聚体（550 kDa）COPI和四聚体（400 kDa）COPII外套的生化解剖，探索外套招募的过程。对于COPI，我们已经建立了6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ARF 1的结构。对于COPII涂层，我们先前确定了与ARF同系物Sar 1结合的涂层内壳的晶体结构。我们现在已经用生物化学方法解剖了COPII外壳，并将确定核心外壳复合物的晶体结构：高质量的晶体（2.6埃分辨率）已经到手。最后，我们将探讨囊泡外套拆卸的GTP水解反应，触发这一进程的重点。我们先前发现COPI加速ARF 1-ARFGAP复合物中GTP水解300倍或更多。我们将通过对COPI核心在基态和过渡态复合物中与ARF 1-ARFGAP结合的生物化学和晶体学分析来研究这种效应的基础。在COPII系统中，最大速率加速的GTTT需要催化剂的贡献，从内壳和外壳的涂层。我们最近定义了一个组装能够最大速率加速，包括内（Sec 23）和外（Sec 31片段）壳分子与Sar 1 GTdR复合。我们已经获得了这种复合物的小晶体，其内部分辨率至少为2.8埃，我们正在努力改进。这些研究将提供深入了解囊泡外壳复合物的GTP依赖性组装和外壳控制的GTP水解触发拆卸的过程。

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.