Structural basis and evolutionary origin of actin filament capping by twinfilin

doi:10.1073/pnas.0608725104

Structural basis and evolutionary origin of actin filament capping by twinfilin

Programs in *Cellular Biotechnology and
^‡Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, FI-00014, Finland; and
^†Cytoskeleton Dynamics and Motility, Laboratoire d'Enzymologie et Biochimie Structurale, Centre National de la Recherche Scientifique, 91118 Gif-sur-Yvette, France

Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved December 16, 2006 (received for review October 3, 2006)

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.

Footnotes

^§To whom correspondence may be addressed at: Institute of Biotechnology, P.O. Box 56, University of Helsinki, FI-00014 Helsinki, Finland. E-mail: pekka.lappalainen{at}helsinki.fi or perttu.permi{at}helsinki.fi
Author contributions: V.O.P. and M.H. contributed equally to this work; V.O.P., M.H., E.H., A.A., M.-F.C., P.P., and P.L. designed research; V.O.P., M.H., E.H., and M.B. performed research; V.O.P. and M.B. contributed new reagents/analytic tools; V.O.P., M.H., E.H., M.B., M.-F.C., P.P., and P.L. analyzed data; and V.O.P., M.H., E.H., A.A., M.-F.C., P.P., and P.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Data deposition: The atomic coordinates of the C-terminal domain of twinfilin have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2HD7).
This article contains supporting information online at www.pnas.org/cgi/content/full/0608725104/DC1.