Fig. 4. The villin headpiece domain of UNC-115 binds to actin filaments. (A) The 639-residue UNC-115 polypeptide. The N terminus consists of three LIM domains and the C-terminus is similar to the headpiece domain of villin (VHD). The region of UNC-115 used in actin-binding assays is indicated by a bar below the diagram. (B) An alignment of the UNC-115 VHD and chicken villin VHD (Gg HP67). Basic residues in the UNC-115 VHD are in blue, and those that were changed to glutamic acid residues to produce the VHD mutant protein are shown with asterisks. The leucine and tryptophan residues that form a hydrophobic `cap' (see below) are boxed. (C) A structural model of the UNC-115 VHD based upon the NMR structure of Gg HP67 (Varder et al., 2002) (see Materials and Methods). Blue, basic groups; red, acidic groups; white, neutral groups. Shown are the `cap' formed by the leucine and tryptophan boxed in B, the charged `crown' (broken line), and the `positive patch' formed by basic residues (Vardar et al., 2002). The locations of basic residues that were changed to glutamic acid residues in UNC-115 VHD mutant protein are indicated by asterisks. (D) A western blot showing that the UNC-115 VHD bound to actin. Equimolar amounts (5 µM) of wild-type and VHD mutant 6HIS::DHFR::UNC-115 (46kD) were mixed with actin filaments, which were then sedimented (see Materials and Methods). Wild-type 6HIS::DHFR::UNC-115 co-sedimented in the presence but not in the absence of actin filaments. Mutant 6HIS::DHFR::UNC-115 (see B) failed to co-sediment with actin filaments.

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