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

Arp2/3复合体是细胞中肌动蛋白的中心成核机器，全长224 kDa，由7种蛋白组成。它控制肌动蛋白丝网络的组装，这对从运动到信号转导到细菌发病等过程至关重要。Arp2/3复合物的内在肌动蛋白成核活性较低，但通过与许多蛋白结合，包括WASp、N-WASp、Scar/WAVE、Bee1和ActA，其成核活性大大增强。Rosen提案的研究重点是发现激活蛋白结合Arp2/3复合物和G-actin以控制组装活性的残基和结构。这项工作扩展了Rosen， Pollard和Li团队之间已有的多年合作。共振核磁共振分析

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.