title: The C-terminal tail of UNC-60B (actin depolymerizing factor/cofilin) is critical for maintaining its stable association with F-actin and is implicated in the second actin-binding site.
abstract: Actin depolymerizing factor (ADF)/cofilin changes the twist of actin filaments by binding two longitudinally associated actin subunits. In the absence of an atomic model of the ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin that are essential for filament binding. Here, we have characterized the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a novel determinant for its association with F-actin. Removal of the C-terminal isoleucine (Ile152) by carboxypeptidase A or truncation by mutagenesis eliminated F-actin binding activity but strongly enhanced actin depolymerizing activity. Replacement of Ile152 by Ala had a similar but less marked effect; F-actin binding was weakened and depolymerizing activity slightly enhanced. Truncation of both Arg151 and Ile152 or replacement of Arg151 with Ala also abolished F-actin binding and enhanced depolymerizing activity. Loss of F-actin binding in these mutants was accompanied by loss or greatly decreased severing activity. All of the variants of UNC-60B interacted with G-actin in an indistinguishable manner from wild type. Cryoelectron microscopy showed that UNC-60B changed the twist of F-actin to a similar extent to vertebrate ADF/cofilins. Helical reconstruction and structural modeling of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might be involved in one of the two actin-binding sites.
authors.affiliation: Department of Pathology, Emory University, Atlanta, Georgia 30322, USA. ono@bimcore.emory.edu
authors.forename: S
authors.initials: S
authors.lastname: Ono
authors.forename: A
authors.initials: A
authors.lastname: McGough
authors.forename: B J
authors.initials: BJ
authors.lastname: Pope
authors.forename: V T
authors.initials: VT
authors.lastname: Tolbert
authors.forename: A
authors.initials: A
authors.lastname: Bui
authors.forename: J
authors.initials: J
authors.lastname: Pohl
authors.forename: G M
authors.initials: GM
authors.lastname: Benian
authors.forename: K M
authors.initials: KM
authors.lastname: Gernert
authors.forename: A G
authors.initials: AG
authors.lastname: Weeds
mesh_terms: D051339:Actin Depolymerizing Factors; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D029742:Caenorhabditis elegans Proteins; D051345:Destrin; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D029742:Caenorhabditis elegans Proteins; D051345:Destrin; D015801:Helminth Proteins; D008840:Microfilament Proteins; C495209:UNC-60 protein, C elegans; D007532:Isoleucine