Analysis of the alpha-actinin/zyxin interaction

The yeast two-hybrid system was used to search for interaction partners of human zyxin. Screening of two different cDNA libraries, one prepared from h uman placenta, the other from human heart, yielded several positive clones that occurred in both searches, including clones coding for cyclophilin, ne bulette, and a-actinin. The zyxin/alpha -actinin interaction was analyzed i n detail. By site-directed mutagenesis, a linear motif of 6 amino acids (Ph e-Gly-Pro-Val-Val-Ala) present at the N terminus of zyxin was found to play a critical role. Replacement of a single amino acid within this motif abol ished binding to a-actinin in blot overlays as well as in living cells. On the other hand, the interaction site in a-actinin was mapped to a conformat ional. determinant present in the center of the protein as demonstrated by a fragment deletion analysis. This binding site involved a tandem array of two complete spectrin-like domains. Only fragments that were able to dimeri ze in yeast also bound to zyxin, suggesting that dimerization of ix-actinin is essential for zyxin binding.