title: Identification of the beta1-integrin binding site on alpha-actinin by cryoelectron microscopy.
abstract: 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.
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA.
authors.forename: Deborah F
authors.initials: DF
authors.lastname: Kelly
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Taylor
mesh_terms: D000185:Actinin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D020285:Cryoelectron Microscopy; D006046:Gold / Q000737:chemistry; D019012:Integrin beta1 / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D008958:Models, Molecular; D049329:Nanostructures; D011485:Protein Binding; D011487:Protein Conformation; D016608:Talin / Q000737:chemistry / Q000378:metabolism
chemical_list: D019012:Integrin beta1; D016608:Talin; D000185:Actinin; D006046:Gold