Association of structural repeats in the alpha-actinin rod domain. Alignment of inter-subunit interactions.
Fragments of the rod domain of chicken alpha-actinin, which comprises four spectrin-like repeat sequences, have been prepared by expression in Escherichia coli. Electron microscopy reveals that all products containing three or four complete repeats are rod-like. Self-association of fragments was detected by chemical cross-linking and analytical equilibrium sedimentation. The intact rod domain forms a stable dimmer, which does not dissociate measurably in the accessible concentration range. Elimination of either terminal repeat (repeat 1 or repeat 4) greatly diminishes 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 extent, but also forms aggregates at high concentrations. The results are most 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 structure, in which one of the terminal repeats does not participate in dimerisation. Possible explanations for the apparent difference observed between the alpha-actinin rod structure in solution and in two-dimensional crystalline arrays are examined.
Flood
G
G
Department of Biochemistry, University of Leicester, UK.
Kahana
E
E
Gilmore
A P
AP
Rowe
A J
AJ
Gratzer
W B
WB
Critchley
D R
DR
D000185:Actinin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003432:Cross-Linking Reagents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013049:Spectrin / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D010455:Peptides; D011994:Recombinant Proteins; D000185:Actinin; D013049:Spectrin; D014357:Trypsin