Structural Dynamics of the Actin-Binding Domains in Dystrophin and Utrophin

Abstract

We are using time-resolved phosphorescence anisotropy (TPA) and dipolar electron-electron-resonance (DEER) to determine and compare the structural dynamics of the actin-binding domains in dystrophin and utrophin. Dystrophin and utrophin bind actin in vitro with similar affinities, but with different molecular contacts (Rybakova et al, 2006, J. Biol. Chem). We hypothesize that these differences alter the elasticity of dystrophin-actin and utrophin-actin linkages to the sarcolemma, affecting the cell's response to muscle stretches, with important implications for muscular dystrophy and its therapy. Our previous TPA studies, detecting the microsecond dynamics of phosphorescent-labeled actin, showed that both proteins have novel effects on actin flexibility, with utrophin more effective than dystrophin (Prochniewicz et al., 2009, PNAS). We have now compared the effects of the isolated actin-binding domains of dystrophin, ABD1 and ABD2. TPA shows that the enhanced rate of actin rotational dynamics is induced primarily by ABD1, while both ABD1 and ABD2 contribute to the restriction in rotational amplitude. Disease-causing point mutations in ABD1 decrease the effects on actin's rotational rate. We propose that this in turn causes the dystrophin-actin complex to be less resilient and thus less able to prevent damage to the muscle cytoskeleton during contraction. Finally, we have attached probes directly to ABD1 in dystrophin and utrophin, to detect changes in structure upon actin binding. High-resolution distance measurements, provided by DEER, show that the two lobes (calponin-homology domains) within ABD1 undergo a dramatic opening upon actin binding, helping to resolve a previous controversy (Lin et al., 2011, PNAS). Analogous studies with dystrophin show more subtle changes upon actin binding, providing insight into the structural and functional differences in dystrophin and utrophin.