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

Arp2/3复合体是细胞内肌动蛋白核化的中心机器，由7种蛋白质组成，大小为224 kDa。它控制着肌动蛋白细丝网络的组装，而肌动蛋白细丝网络是从运动到信号转导到细菌发病过程中必不可少的过程。Arp2/3复合体固有的肌动蛋白成核活性很低，但通过与许多蛋白质的结合，包括黄蜂、N-黄蜂、SCAR/WAVE、Bee1和ActA，Arp2/3复合体的肌动蛋白成核活性大大增强。Rosen建议的研究重点是发现激活蛋白通过与Arp2/3复合体和G-肌动蛋白结合来控制组装活性的残基和结构。这项工作延续了罗森、波拉德和李团队之间现有的多年合作。共振的核磁共振分析 拓宽和转移NOE将迅速发现与Arp2/3复合体接触的一系列激活子的VCA片段中的特定区域和结构。突变实验将确定单个接触对Arp2t3复合体结合和激活的热力学贡献，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耦合是由C区还是A区介导的。核磁共振和诱变研究将测试全长黄蜂/N-黄蜂与Arp2/3复合体相互作用的两个相互竞争的模型，以发现这些蛋白质的B和GBD区域是通过直接与组装结合来调节Arp2/3复合体的活性，还是间接通过调节VCA的亲和力来调节Arp2/3复合体的活性。对选择性标记的完整Arp2/3复合体的核磁共振研究将揭示介导与激活剂结合的ARPC3/p21和ARPC5/p16亚单位的表面，并将导致用核磁共振对超大型系统进行机械分析的一般程序。最后，核磁共振和诱变实验将确定VCA多肽与G-肌动蛋白结合的结构和热力学基础。合并后的数据将使我们能够辨别Arp2/3复合激活剂之间的共同特征和关键差异，并开发出活化的结构和热力学模型。整个实验将只会是 可能通过罗森、波拉德和李小组之间的试剂和专业知识的合作交流。识别VCA片段和全长黄蜂中最小的Arp2/3复杂结合和激活元件将指导Pollard/Almo和Hanein/Volkmann提案中的结晶学、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.