Dystrophin is an actin binding protein that is thought to stabilize the cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule [from N-terminal actin binding domain (ABD) 1 through ABD2] bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays than full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction by restricting actin rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin.
Bibliographical noteFunding Information:
We thank Bin Li for excellent technical support. This work was supported by the National Institutes of Health (NIH) Training Program in Muscle Research ( AR007612 ), NIH grant RO1 AR042423 to J.M.E., NIH grant R01 AG026160 to D.D.T., and NIH grant F30 AG034033 to A.Y.L. TPA experiments were performed in the Biophysical Spectroscopy Facility supported by NIH grant P30 AR057220 .
- actin binding protein
- actin dynamics
- cooperative binding
- muscular dystrophy