title: Large-scale opening of utrophin's tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism.
abstract: We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin-actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron-electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Ava Y
authors.initials: AY
authors.lastname: Lin
authors.forename: Ewa
authors.initials: E
authors.lastname: Prochniewicz
authors.forename: Zachary M
authors.initials: ZM
authors.lastname: James
authors.forename: Bengt
authors.initials: B
authors.lastname: Svensson
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D049411:Utrophin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D011994:Recombinant Proteins; D049411:Utrophin