title: Structural basis and evolutionary origin of actin filament capping by twinfilin.
abstract: Dynamic reorganization of the actin cytoskeleton is essential for motile and morphological processes in all eukaryotic cells. One highly conserved protein that regulates actin dynamics is twinfilin, which both sequesters actin monomers and caps actin filament barbed ends. Twinfilin is composed of two ADF/cofilin-like domains, Twf-N and Twf-C. Here, we reveal by systematic domain-swapping/inactivation analysis that the two functional ADF-H domains of twinfilin are required for barbed-end capping and that Twf-C plays a critical role in this process. However, these domains are not functionally equivalent. NMR-structure and mutagenesis analyses, together with biochemical and motility assays showed that Twf-C, in addition to its binding to G-actin, interacts with the sides of actin filaments like ADF/cofilins, whereas Twf-N binds only G-actin. Our results indicate that during filament barbed-end capping, Twf-N interacts with the terminal actin subunit, whereas Twf-C binds between two adjacent subunits at the side of the filament. Thus, the domain requirement for actin filament capping by twinfilin is remarkably similar to that of gelsolin family proteins, suggesting the existence of a general barbed-end capping mechanism. Furthermore, we demonstrate that a synthetic protein consisting of duplicated ADF/cofilin domains caps actin filament barbed ends, providing evidence that the barbed-end capping activity of twinfilin arose through a duplication of an ancient ADF/cofilin-like domain.
authors.affiliation: Program in Cellular Biotechnology and Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, FI-00014, Finland.
authors.forename: Ville O
authors.initials: VO
authors.lastname: Paavilainen
authors.forename: Maarit
authors.initials: M
authors.lastname: Hellman
authors.forename: Emmanuèle
authors.initials: E
authors.lastname: Helfer
authors.forename: Miia
authors.initials: M
authors.lastname: Bovellan
authors.forename: Arto
authors.initials: A
authors.lastname: Annila
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Carlier
authors.forename: Perttu
authors.initials: P
authors.lastname: Permi
authors.forename: Pekka
authors.initials: P
authors.lastname: Lappalainen
mesh_terms: D051344:Actin Capping Proteins / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D019143:Evolution, Molecular; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary
chemical_list: D051344:Actin Capping Proteins; D000199:Actins; D029721:Drosophila Proteins; D008840:Microfilament Proteins; C442839:twf protein, Drosophila