Identification of the β1-integrin binding site on α-actinin by cryoelectron microscopy

Cell-matrix adhesions in migrating cells are usually mediated by integrins, α-β heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the β1-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 α-actinin. The 2-D arrays of the β1-integrin-α-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 β1-integrin peptide. The difference maps indicate that the β1-integrin cytoplasmic domain binds α-actinin between the first and second, 3-helix motifs in the central rod domain.