Structure and function of Arp 2/3 complex--Subproject 2

Arp 2/3复合物是由7种蛋白质组成的分子量为224 kDa的复合体，是细胞内的核心肌动蛋白成核机。它控制肌动蛋白丝网络的组装，这些网络对从运动到信号转导到细菌致病的过程至关重要。Arp 2/3复合物的内在肌动蛋白成核活性较低，但通过与许多蛋白质（包括WASp、N-WASp、Scar/WAVE、Bee 1和ActA）结合而大大增强。罗森提案的研究重点是发现激活蛋白通过Arp 2/3复合物和G-肌动蛋白结合来控制组装体活性的残基和结构。这项工作扩展了罗森、波拉德和李的团队之间现有的多年合作。共振的NMR分析 加宽和转移的NOE将迅速发现与Arp 2/3复合物接触的一系列激活剂的VCA片段内的特定区域和结构。诱变实验将确定单个接触对Arp 2 t3复合物结合和活化的热力学贡献，并将发现VCA结合与F-肌动蛋白、核苷酸或G-肌动蛋白结合的热力学偶联是否由C或A区介导。NMR和诱变研究将测试全长WASp/N-WASp与Arp 2/3复合物相互作用的两种竞争模型，以发现这些蛋白质的B和GBD区域是否通过直接结合到组装体或间接通过调节VCA的亲和力来调节Arp 2/3复合物的活性。选择性标记的完整Arp 2/3复合物的NMR研究将揭示介导与活化剂结合的ARPC 3/p21和ARPC 5/p16亚基的表面，并将导致通过NMR对非常大的系统进行机械分析的一般程序。最后，NMR和诱变实验将定义VCA肽与G-肌动蛋白结合的结构和热力学基础。合并后的数据将允许Arp 2/3复合物激活剂之间的共同特点和关键差异被识别，并开发激活的结构和热力学模型。整个实验过程中， 通过罗森、波拉德和李三家集团之间的试剂和专门知识的合作交流，这是可能的。VCA片段和全长WASp中最小Arp 2/3复合物结合和激活元件的鉴定将指导Pollard/Almo和Hanein/Bermmann提案中的晶体学、辐射足迹和EM研究，并有助于解释其中确定的结构。

Arp2/3 complex, a 224 kDa assembly of 7 proteins, is the central actin nucleation machine in the cell. It controls assembly of actin filament networks that are essential to process ranging from motility to signal transduction to bacterial pathogenesis. The intrinsic actin nucleating activity of Arp2/3 complex is low, but is greatly enhanced through binding of many proteins, including WASp, N-WASp, Scar/WAVE, Bee1 and ActA. Research in the Rosen Proposal is focused on discovering the residues and structures through which activator proteins bind Arp2/3 complex and G-actin to control activity of the assembly. The work extends an existing multi-year collaboration between the Rosen, Pollard and Li groups. NMR analyses of resonance broadening and transferred-NOEs will rapidly discover the specific regions and structures within the VCA segments of a series of activators that contact Arp2/3 complex. Mutagenesis experiments will define the thermodynamic contributions of individual contacts to binding and activation of Arp2t3 complex, and will discover if thermodynamic coupling of VCA binding to binding of F-actin, nucleotides or G-actin is mediated by the C or A regions. NMR and mutagenesis studies will test two rival models for the interaction of full length WASp/N-WASp with Arp2/3 complex to discover whether the B and GBD regions of these proteins modulate activity of Arp2/3 complex through direct binding to the assembly, or indirectly through modulating affinity of the VCA. NMR studies of selectively labeled intact Arp2/3 complex will reveal the surfaces of the ARPC3/p21 and ARPC5/p16 subunits that mediate binding to activators, and will lead to general procedures for mechanistic analysis of very large systems by NMR. Finally, NMR and mutagenesis experiments will define the structural and thermodynamic bases for binding of VCA peptides to G-actin. The combined data will allow common features and key differences among Arp2/3 complex activators to be discerned, and structural and thermodynamic models of activation to be developed. Experiments throughout will only be possible through collaborative exchange of reagents and expertise among the Rosen, Pollard and Li groups. Identification of minimal Arp2/3 complex binding and activation elements in VCA segments and full length WASp will direct crystallographic, radiation footprinting and EM studies in the Pollard/Almo and Hanein/Volkmann Proposals, and aid interpretation of structures determined therein.