The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers.
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) = 0.05 microM) than ATP-G-actin (K(D) = 0.47 microM) under physiological ionic conditions and forms a relatively stable (k(off) = 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.
Ojala
Pauli J
PJ
Program of Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland.
Paavilainen
Ville O
VO
Vartiainen
Maria K
MK
Tuma
Roman
R
Weeds
Alan G
AG
Lappalainen
Pekka
P
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D046911:Macromolecular Substances; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D051339:Actin Depolymerizing Factors; D000199:Actins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C485190:Twf1 protein, mouse