title: Structure of a longitudinal actin dimer assembled by tandem w domains: implications for actin filament nucleation.
abstract: Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.
authors.affiliation: Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Rebowski
authors.affiliation: Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
authors.forename: Suk
authors.initials: S
authors.lastname: Namgoong
authors.affiliation: Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Boczkowska
authors.affiliation: Boston Biomedical Research Institute, Watertown, MA 02472-2899, USA.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Leavis
authors.affiliation: Institut de Biologie Structurale, F-38027 Grenoble, France.
authors.forename: Jorge
authors.initials: J
authors.lastname: Navaza
authors.affiliation: Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA. Electronic address: droberto@mail.med.upenn.edu.
authors.forename: Roberto
authors.initials: R
authors.lastname: Dominguez
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011817:Rabbits; D014736:Vibrio parahaemolyticus / Q000737:chemistry
chemical_list: D000199:Actins; D001426:Bacterial Proteins
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