Structural basis for Rab GTPase regulated membrane trafficking

描述（由申请人提供）：Rab GTP酶是真核细胞内膜系统中膜成熟和运输的关键主调节因子。Rab GTP酶的功能取决于“非活性”（GDP结合）和“活性”（GTP结合）状态之间的构象转换循环。激活和失活由GDP/GTP交换因子（GEF）和GTP酶激活蛋白（GAP）严格控制，其加速核苷酸交换和GTP水解的固有缓慢速率。活性Rab GTP酶与参与膜运输的所有阶段的不同效应物相互作用。活性Rab GTP酶的功能输出通过具有多个Rab结合位点的多价效应物整合。活性Rab GTP酶还为下游或上游Rab GTP酶募集GEF或GAP，以协调连续的运输阶段并促进膜成熟。本提案的总体目标是了解Rab GEF和GAP的膜募集、募集后Rab GTP酶的选择性激活和失活以及微生物病原体对这些宿主过程的操纵的结构和分子基础。具体而言，我们将联合收割机结合晶体学、突变和基于细胞的分析与定量Rab家族识别谱，以研究以下的结构和分子基础：（目的1）DENN和Vps 9结构域GEF的募集和Rab识别/激活;（目的2）TBC结构域GAP的募集和Rab识别/激活;（目的3）嗜肺军团菌对宿主Rab GTP酶的操纵。这些目标的成功完成将提供新的见解，以支持在正常和致病条件下的Rab GTP酶调节膜生物学的分子相互作用网络和结构机制。

DESCRIPTION (provided by applicant): Rab GTPases are key master regulators of membrane maturation and trafficking throughout the endomembrane system of eukaryotic cells. The function of Rab GTPases depends on a conformational switch cycle between 'inactive' (GDP-bound) and 'active' (GTP-bound) states. Activation and deactivation are tightly controlled by GDP/GTP exchange factors (GEFs) and GTPase activating proteins (GAPs) that accelerate the intrinsically slow rates of nucleotide exchange and GTP hydrolysis. Active Rab GTPases interact with diverse effectors involved in all stages of membrane trafficking. The functional outputs of active Rab GTPases are integrated through multivalent effectors with multiple Rab binding sites. Active Rab GTPases also recruit GEFs or GAPs for downstream or upstream Rab GTPases to coordinate successive trafficking stages and facilitate membrane maturation. The overall goal of this proposal is to understand the structural and molecular bases underlying membrane recruitment of Rab GEFs and GAPs, selective activation and deactivation of Rab GTPases following recruitment, and manipulation of these host process by microbial pathogens. Specifically, we will combine crystallographic, mutational and cell based analyses with quantitative Rab family recognition profiles to investigate the structural and molecular bases for: (Aim 1) recruitment and Rab recognition/activation by DENN and Vps9 domain GEFs; (Aim 2) recruitment and Rab recognition/activation by TBC domain GAPs; (Aim 3) manipulation of host Rab GTPases by Legionella pneumophila. Successful completion of these aims will deliver new insights into poorly characterized molecular interaction networks and structural mechanisms that underpin Rab GTPase-regulated membrane biology in normal and pathogenic conditions.