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A more recent version of this article appeared on November 1, 2002
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Submitted on March 18, 2002
Revised on August 15, 2002
Accepted on September 4, 2002
1 Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014, Helsinki, Finland
2 Structural Biology, Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014, Helsinki, Finland
3 MRC Laboratory of Molecular Biology, Cambridge, CB2 2QH, England
* Corresponding author. E-mail address: pekka.lappalainen{at}helsinki.fi.
Twinfilin is a ubiquitous and abundant actin monomer-binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (KD=0.05 µM) than ATP-G-actin (KD=0.47 µM) under physiological ionic conditions, and forms a relatively stable (koff=1.8 s-1) complex with ADP-G-actin. Data from native polyacrylamide gel electrophoresis and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations twinfilin, cofilin and actin may also form a ternary complex. By systematic deletion analysis we show that the actin-binding activity is located entirely in twinfilin's two ADF-H domains. Individually these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has more than 10-fold higher affinity for ADP-G-actin, is almost entirely responsible for twinfilin's ability to increase the amount of monomeric actin in co-sedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. Based on these results we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.
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