Analysis of the subunit interactions in alpha-Actinin

Recombinant fragments of the chicken smooth muscle -actinin rod domain, which contains 4 spectrin-like repeats, were used in an endeavour to discriminate between aligned and staggered models of the -actinin homodimer. The results indicate that deletion of either of the terminal repeats, 1 or 4, drastically reduces the dimer formation, and an aligned rather than a staggered model most accurately explains these observations. This model was further confirmed by the demonstration of an interaction between the individual terminal repeats I and 4, which in a staggered model would seem to be futile. Mutation of a conserved tryptophan in repeat 1 of a polypeptide spanning the -actinin rod domain resulted in a decrease in dimer stability compared to the native rod, although dissociation of the dimer still required denaturing conditions. When the effect of this mutation was examined in the isolated single repeat 1, a significant change in solubility was observed, and assumed to reflect a more drastic conformational alteration. A similar result was obtained when a predicted 8-residue insertion was deleted from the N-terminal region of the same repeat. A deletion of a predicted 8-residue insertion from the N-terminal region of repeat 4 also affected conformational stability, and eliminated the heterologous interaction with native repeat 1. However, when the predicted 8-residue insert in repeat 1 was deleted, in a polypeptide spanning the entire rod domain, no decrease in stability or dimer-forming ability was detected.