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Originally published as MBC in Press, 10.1091/mbc.E02-03-0157 on September 24, 2002
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Vol. 13, Issue 11, 3811-3821, November 2002

The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

Pauli J. Ojala,* Ville O. Paavilainen,* Maria K. Vartiainen,* Roman Tuma,dagger Alan G. Weeds,Dagger and Pekka Lappalainen*§

Programs in  *Cellular Biotechnology and  dagger Structural Biology, Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland; and  Dagger MRC Laboratory of Molecular Biology, Cambridge, CB2 2QH, England

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 PAGE 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 the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation 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 a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

§ Corresponding author. E-mail address: pekka.lappalainen{at}helsinki.fi.

Molecular Biology of the Cell
Vol. 13, 3811-3821, November 2002
Copyright © 2002 by The American Society for Cell Biology


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