Structure and Function of Arp 2/3 Complex

Arp2/3复合体是一个进化上保守的7个亚基组合，在细胞运动中起关键作用。在细胞运动过程中，它响应各种信号介导分支肌动蛋白丝网络的组装。我们提出了一系列互补的方法来了解核苷酸和激活肽如何影响Arp2/3复合物的构象以及Arp2/3复合物如何形成肌动蛋白丝分支。(1) Pollard和Almo实验室将利用x射线晶体学和辐射足迹技术提供Arp2/3复合物活性构象的高分辨率结构，以记录激活该复合物使肌动蛋白丝成核所需的构象变化，并绘制蛋白质之间的相互作用图

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.