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

Abstract

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 (K D ϭ 0.05 M) than ATP-G-actin (K D ϭ 0.47 M) under physiological ionic conditions and forms a relatively stable (k off ϭ 1.8 s Ϫ1 ) complex with ADP-Gactin. 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 Ͼ10-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.

TetraThymosinβ Is Required for Actin Dynamics inCaenorhabditis elegansand Acts via Functionally Different Actin-binding Repeats

“…Gelsolin is a well-known example where homologous segments have different actin-binding activities and their combined activity is responsible for severing and subsequent capping (Pope et al, 1991;Van Troys et al, 1996a). Multiple differential actin-binding units have also been reported in Ena/VASP proteins (Bachmann et al, 1999;Walders-Harbeck et al, 2002), Calponin Homology-domain containing proteins (Gimona et al, 2002), twinfilin (Ojala et al, 2002), MARCKS (Yarmola et al, 2001), and MIM (Mattila et al, 2003;Yamagishi et al, 2004).…”

Structural Conservation between the Actin Monomer-binding Sites of Twinfilin and Actin-depolymerizing Factor (ADF)/Cofilin

“…The proteins were cleaved from GST by thrombin digestion and purified by gel filtration chromatography as described previously (10). The site-directed mutations were introduced to the Twf 1-174 construct using the PCR-based overlap extension method described by Higuchi et al (18).…”

“…Actin Monomer Binding Assay-The change in the fluorescence of NBD-labeled ADP-G-actin was used to monitor the binding of wild-type and mutant twinfilin domains to actin monomers as described (10). ADP-actin was prepared by incubating NBD-actin with hexokinaseagarose beads (Sigma) and 1 mM glucose overnight as described (30).…”

“…Unlike ADF/cofilins and Abp1/drebrins, which interact with actin filaments, twinfilins bind only actin monomers (9). Twinfilins bind ADP-G-actin with ϳ10-fold higher affinity than ATP-G-actin and prevent the nucleotide exchange on actin monomers (10). Analysis of yeast twinfilin suggested that this protein contributes to actin dynamics by localizing actin monomers, in their "inactive" ADP-form, to the sites of rapid actin assembly in cells.…”

“…Kinetic analysis further suggests that the actin monomer first associates with the N-terminal ADF-H domain and is then delivered to the C-terminal domain through a conformational change within the twinfilin molecule (10). The atomic structures of four ADF/cofilin proteins have been determined by x-ray crystallography and NMR (13)(14)(15)(16).…”