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

• DOI: 10.1091/mbc.e02-03-0157
• Publication Date: 2002-11-12T19:42:39Z
• Authors: Pauli J. Ojala; Ville O. Paavilainen; Maria K. Vartiainen; Roman Tuma; Alan G. Weeds; Pekka Lappalainen
• Keywords: 0303 health sciences; Binding Sites; Macromolecular Substances; Protein Conformation; Recombinant Fusion Proteins; Microfilament Proteins; Molecular Sequence Data; Models, Biological; Actins; Adenosine Diphosphate; Mice; 03 medical and health sciences; Adenosine Triphosphate; Actin Depolymerizing Factors; Animals; Amino Acid Sequence; Sequence Alignment; Protein Binding

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.