Mechanistic Aspects of Actin Polymerization at WH2-proteins: Human Spir and VopF from V. cholerae in Comparison

肌动蛋白的成核和聚合需要不同类型的蛋白质支架，这些支架可以隔离和定位游离的肌动蛋白分子。Arp2/3复合体已被报道聚合支化的肌动蛋白细丝，而已知的Forin样核剂在带刺的末端形成未支化的肌动蛋白聚合物。第三个因子Spir被描述为通过四个随后的WASP同源2结构域(WH2)的出现从尖端启动未分支的肌动蛋白细丝。由于Spir定位于膜表面，该蛋白可能在细胞内的囊泡运输中发挥作用。Spir序列包括四个结构域，Kind结构域、WH2簇、Spir-box和FYVE结构域，用于膜相互作用。Spir与膜的结合和肌动蛋白聚合过程的启动涉及许多辅助因素：肌动蛋白单体、Forin，可能还有膜结合的GTP酶。为了从机理上理解Spir对成核和聚合的调节，我们已经开始用结构生物学的方法来绘制相互作用图。我们的第一个尝试是确定Spir/Kind和Forin Spir相互作用(FSI)模块之间的复杂结构。最近发现，致病细菌也使用分泌的效应蛋白中的WH2结构域簇来操纵宿主细胞的细胞骨架。我们目前正在研究霍乱弧菌VopF的结构，模拟Spir蛋白的WH2-支架。在这里，我们的目的是了解对宿主细胞途径的干扰，并总体上比较WH2结构域簇上肌动蛋白细丝形成的原理。

Initiation of actin nucleation and polymerization requires protein scaffolds of different types which allow for sequestration and orientation of free actin molecules. The Arp2/3 complex has been reported to polymerize branched actin filaments, while formin-like nucleators are known to form unbranched actin polymers at the barbed end. A third factor, Spir, has been described to initiate unbranched actin filaments from the pointed end by the occurrence of four subsequent WASP Homology 2 domains (WH2). Due to the localization of Spir at membrane surfaces, a possible function of this protein in vesicle transport within cells has been proposed. The Spir sequence comprises four domains, the KIND domain, the WH2 cluster, the Spir-box and the FYVE domain for membrane interaction. Attachment of Spir to membranes and initiation of actin polymerization processes involves a number of co-factors: actin monomers, formin, and possibly membrane-bound GTPases. To understand the regulation of nucleation and polymerization by Spir on a mechanistic basis we have started to map interactions by structure biology methods. Our first attempt was the determination of a complex structure between the Spir/KIND and the formin Spir interaction (FSI) module. More recently it turned out that also pathogenic bacteria use WH2- domain clusters in secreted effector proteins to manipulate the host cell cytoskeleton. We currently investigate the structure of VopF from Vibrio cholerae mimicking the WH2-scaffold of Spir proteins. Here we aim to understand the interference into the host cell pathways and to compare principles of actin filament formation on WH2 domain clusters in general.