D. Hanein, L.E. Rost, P. Matsudaira* and D.J. DeRosier
Rosenstiel Basic Medical Sciences Research Center, Brandeis, University, Waltham, MA 02254 ; and *Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142.
The state of actin is a function of its association with actin binding proteins. Actin binding proteins are often bivalent molecules that can crosslink actin to form gels or bundles in vitro and in vivo. The actin bundles found in the microvilli of the intestinal brush border contain the bundling proteins fimbrin and villin. Fimbrin belongs to a superfamily of actin cross-linking proteins that share homologous 27-kDa actin-binding domains. The three-dimensional structures of actin filaments and of actin filaments decorated with fimbrin (N375) were determined to 28Å resolution using electron cryo-microscopy and image analysis. In the difference maps between the two structures, N375 is seen to contact a concave surface of the actin filament formed by subdomains 1 and 2 of one actin monomer and subdomain 1 of the neighboring monomer along the long-pitch helix. The contact region includes actin residues 146-148 and 350-355 from one monomer, and 43-100 on the adjacent monomer. Moreover, N375 binding is accompanied by a conformational change in actin which appears to be a shift in subdomain 1. This is a novel conformational change in actin, and it shows that actin binding proteins do modulate the structure of actin and therefore can alter its functional properties. Villin belongs to a different class of proteins, which can bind and sever actin filaments. Unlike other members of its class, villin has a second and unique actin binding domain, which enables it to bundle actin filaments. This second actin binding domain termed villin headpiece, is a 76 residue region at the C terminal end of the molecule. In three-dimensional reconstructions from electron micrographs, the villin headpiece appears to bind away from the fimbrin (N375) site and near the phalloidin site on actin.