IDENTIFICATION OF THE VINCULIN-BINDING SITE IN THE CYTOSKELETAL PROTEIN ALPHA-ACTININ

Using low-speed sedimentation equilibrium we have established that vin culin binds to alpha-actinin with a K-d of 1.3 x 10(-5) M. Electron mi croscopy of negatively stained preparations of vinculin revealed spher ical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha- actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both 'lollipop'- and ' dumbell'-shaped particles which we interpret as either one or two sphe rical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actin in using I-125-vinculin and a gel-blot assay in which proteolytic frag ments of alpha-actinin and fragments of alpha-actinin expressed in Esc herichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. I-125- vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-ter minal end of the molecule (alpha-actinin residues 713-749). I-125-vinc ulin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding si te is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.