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.
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Authors | Ava Y Lin, Ewa Prochniewicz, Zachary M James, Bengt Svensson, David D Thomas |
Journal | Proceedings of the National Academy of Sciences of the United States of America
(Proc Natl Acad Sci U S A)
Vol. 108
Issue 31
Pg. 12729-33
(Aug 02 2011)
ISSN: 1091-6490 [Electronic] United States |
PMID | 21768337
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
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Chemical References |
- Actins
- Recombinant Proteins
- Utrophin
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Topics |
- Actins
(chemistry, metabolism)
- Animals
- Binding Sites
(genetics)
- Binding, Competitive
- Circular Dichroism
- Electron Spin Resonance Spectroscopy
- Kinetics
- Mice
- Microscopy, Electron
- Models, Molecular
- Mutation
- Protein Binding
- Protein Conformation
- Protein Structure, Secondary
- Protein Structure, Tertiary
- Recombinant Proteins
(chemistry, metabolism)
- Utrophin
(chemistry, genetics, metabolism)
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