Determination of the α-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis

Abstract

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle α-actinin (αA1-2) has been determined to 21-Å resolution. The shape and location of αA1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. αA1-2 resembles a bell that measures ~38 Å at its base and extends 42 Å from its base to its tip. In decorated filaments, the base of αA1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two- start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with αA1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for α- actinin on adjacent actin monomers.