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

肌动蛋白成核和聚合的启动需要不同类型的蛋白质支架，这些支架允许游离肌动蛋白分子的隔离和定向。据报道，Arp2/3复合物可聚合支链肌动蛋白细丝，而已知形成蛋白样核子可在有刺端形成未支链肌动蛋白聚合物。第三个因子Spir已被描述为通过四个后续的WASP同源2结构域（WH2）的发生，从尖端启动未分支的肌动蛋白细丝。由于Spir在膜表面的定位，该蛋白可能在细胞内的囊泡运输中起作用。Spir序列包括4个结构域：KIND结构域、WH2簇结构域、Spir-box结构域和用于膜相互作用的FYVE结构域。Spir附着在膜上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、双胍和可能的膜结合gtp酶。为了在机制基础上理解Spir对成核和聚合的调控，我们已经开始用结构生物学方法绘制相互作用图。我们的第一次尝试是确定Spir/KIND和formin Spir相互作用（FSI）模块之间的复杂结构。最近发现，致病菌也利用分泌效应蛋白中的WH2结构域簇来操纵宿主细胞骨架。我们目前正在研究来自霍乱弧菌的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通路的干扰，并比较一般情况下WH2结构域簇上肌动蛋白丝形成的原理。

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.