title: Three-dimensional reconstructions of Arp2/3 complex with bound nucleation promoting factors.
abstract: Arp2/3 complex initiates the growth of branched actin-filament networks by inducing actin polymerization from the sides of pre-existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three-dimensional structures at ~2 nm resolution of Arp2/3 complex with three different bound NPFs: N-WASp, Scar-VCA and cortactin. All of these structures adopt a conformation with the two actin-related proteins in an actin-filament-like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin-filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.
authors.affiliation: Bioinformatics and Systems Biology Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Xu
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rouiller
authors.forename: Brian D
authors.initials: BD
authors.lastname: Slaughter
authors.forename: Coumaran
authors.initials: C
authors.lastname: Egile
authors.forename: Eldar
authors.initials: E
authors.lastname: Kim
authors.forename: Jay R
authors.initials: JR
authors.lastname: Unruh
authors.forename: Xiaoxue
authors.initials: X
authors.lastname: Fan
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pollard
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Dorit
authors.initials: D
authors.lastname: Hanein
authors.forename: Niels
authors.initials: N
authors.lastname: Volkmann
mesh_terms: D000048:Acanthamoeba; D051377:Actin-Related Protein 2 / Q000737:chemistry*; D051378:Actin-Related Protein 3 / Q000737:chemistry*; D001665:Binding Sites; D005656:Fungal Proteins / Q000737:chemistry*; D008854:Microscopy, Electron; D008958:Models, Molecular; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry*; D004718:Saccharomycetales; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000737:chemistry*
chemical_list: D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D005656:Fungal Proteins; D015800:Protozoan Proteins; D051300:Wiskott-Aldrich Syndrome Protein Family