ASSOCIATION OF STRUCTURAL REPEATS IN THE ALPHA-ACTININ ROD DOMAIN - ALIGNMENT OF INTER-SUBUNIT INTERACTIONS

Fragments of the rod domain of chicken a-actinin, which comprises four spectrin-like repeat sequences, have been prepared by expression in E scherichia coli. Electron microscopy reveals that all products contain ing three or four complete repeats are rod-like. Self-association of f ragments was detected by chemical cross-linking and analytical equilib rium sedimentation. The intact rod domain forms a stable dimer, which does not dissociate measurably in the accessible concentration range. Elimination of either terminal repeat (repeat 1 or repeat 4) greatly d iminishes the extent of dimerisation. The fragment comprising repeats 1-3 dimerises appreciably, with an association constant estimated from the sedimentation equilibrium distribution of approximately 5 x 10(5) M(-1). The fragment made up of repeats 2-4 dimerises to a small exten t, but also forms aggregates at high concentrations. The results are m ost easily reconciled with an aligned structure for the rod domain in solution, in which repeat 1 associates with repeat 4 of the partnering chain, and repeat 2 with repeat 3, rather than with a staggered struc ture, in which one of the terminal repeats does not participate in dim erisation. Possible explanations for the apparent difference observed between the alpha-actinin rod structure in solution and in two-dimensi onal crystalline arrays are examined. (C) 1995 Academic Press Limited