Identification of the beta1-integrin binding site on alpha-actinin by cryoelectron microscopy.

Cell-matrix adhesions in migrating cells are usually mediated by integrins, alpha-beta heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the beta1-integrin (residues H738-K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated-nickel group (dimyristoylphosphatidyl choline-suberimide-nitriloacetic acid:nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. A Nanogold particle was covalently linked to cysteines introduced at the C-terminus and after residue T757 on the integrin peptide, and co-crystallized with chicken smooth muscle alpha-actinin. The 2-D arrays of the beta1-integrin-alpha-actinin complex were examined by cryoelectron microscopy, with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled beta1-integrin peptide. The difference maps indicate that the beta1-integrin cytoplasmic domain binds alpha-actinin between the first and second, 3-helix motifs in the central rod domain.