Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

• Published in: Journal of molecular biology Vol. 403; no. 1; pp. 11 - 23
• Main Authors: Rebowski, Grzegorz, Namgoong, Suk, Boczkowska, Malgorzata, Leavis, Paul C., Navaza, Jorge, Dominguez, Roberto
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 15-10-2010
• Subjects: 60 APPLIED LIFE SCIENCES; ACTIN; Actin Cytoskeleton - metabolism; actin monomer sequestration; actin nucleation; Actins - chemistry; Actins - metabolism; Animals; Bacterial Proteins - chemistry; BASIC BIOLOGICAL SCIENCES; CROSS-LINKING; CRYSTAL STRUCTURE; Crystallography, X-Ray; DIMERS; ELONGATION; Models, Molecular; MONOMERS; NUCLEATION; nucleation-promoting factors; POLYMERIZATION; Protein Multimerization; Protein Structure, Quaternary; Rabbits; Tβ4; Vibrio parahaemolyticus - chemistry; W domain; actin nucleation; NPF; actin monomer sequestration; Tβ4; D-loop; W domain; nucleation-promoting factors; SAXS; PDB
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2010.08.040

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.