Structure and Function of Arp 2/3 Complex

Arp2/3复合体是由7个亚基组成的进化保守的组合体，在细胞运动中起关键作用。作为对各种信号的响应，它在细胞运动过程中介导了分支肌动蛋白细丝网络的组装。我们提出了一系列互补的方法来了解核苷酸和活化肽如何影响Arp2/3复合体的构象，以及Arp2/3复合体如何形成肌动蛋白细丝分支。(1)Pollard和Almo实验室将使用X射线结晶学和辐射足迹法来提供Arp2/3复合体活性构象的高分辨率结构，以记录激活该复合体以使肌动蛋白细丝成核所需的构象变化，以及蛋白质之间的MAP相互作用 分支的组成部分，包括母丝的侧面。(2)罗森实验室将使用核磁共振技术分析激活肽与Arp2/3复合体的结合以及激活肽和核苷酸引起的构象变化。(3)Hanein和Volkmann实验室将使用电子冷冻显微镜和单粒子分析来识别Arp2/3复合体的各种构象状态中的单个亚基与各种结合核苷酸和 激活肽。他们还将重建结合在肌动蛋白细丝一侧的Arp2/3复合体以及由Arp2/3复合体形成的肌动蛋白细丝分支的结构。 (4)LI实验室将制备新形式的酵母Arp2/3复合体，其中包括用于核磁共振的同位素标记的亚基和用于电子显微镜的金标记的标记亚基。LI实验室将使用突变产生具有错误核苷酸结合或水解或其他缺陷的Arp2/3复合体，以捕获有助于分析结构、生化途径和生理机制的构象状态。

Arp2/3 complex is an evolutionarily conserved assembly of seven subunits with a key role in cellular motility. In response to various signals it mediates the assembly of branched actin filament networks during cellular movements. We propose a range of complementary methods to learn how nucleotides and activating peptides influence the conformation of Arp2/3 complex and how Arp2/3 complex forms actin filament branches. (1) The Pollard and Almo labs will use X-ray crystallography and radiation foot printing to provide high resolution structures of active conformations of Arp2/3 complex to document the conformational changes required to activate the complex for nucleating actin filaments and map interactions between the protein components of branches including the side of the mother filament. (2) The Rosen lab will use NMR to analyze activator peptide binding to Arp2/3 complex and conformational changes induced by activator peptides and nucleotides. (3) The Hanein and Volkmann labs will use electron cryomicroscopy and single-particle analysis to identify individual subunits in a range of conformationaI states of Arp2/3 complex with various bound nucleotides and activating peptides. They will also reconstruct Arp2/3 complex bound to the side of an actin filament and the structure of actin filament branches formed by Arp2/3 complex. (4) The Li lab will prepare novel forms of yeast Arp2/3 complex with isotopically labeled subunits for NMR and tagged subunits for labeling with gold for electron microscopy. The Li lab will use mutagenesis to generate Arp2/3 complex with faulty nucleotide binding or hydrolysis or other defects to trap conformational states useful for analysis of structures, biochemical pathways and physiological mechanisms.