Identification of spectrin-like repeats required for high affinity utrophin-actin interaction.
Most studies aimed at characterizing the utrophinactin interaction have focused on the amino-terminal tandem calponin homology domain. However, we recently reported evidence suggesting that spectrin-like repeats of utrophin also participate in binding to actin. Here we expressed several recombinant fragments encoding the utrophin amino-terminal domain alone or in combination with various numbers of spectrin-like repeats. We further quantitatively characterized the actin binding properties of each recombinant utrophin fragment using a high-speed sedimentation assay. To evaluate the capacity of each protein to stabilize actin filaments, we compared the effect of utrophin recombinant fragments and full-length utrophin on 6-propionyl-2-(N,N-dimethylamino)naphthalene actin depolymerization. Our results suggest that, whereas the amino-terminal domain is essential for primary interaction between utrophin and actin, spectrin-like repeats have additive effects on the affinity and stoichiometry of binding. Our data indicate that the amino-terminal domain and first 10 consecutive spectrin-like repeats recapitulate the actin binding activity of full-length utrophin more faithfully than the amino-terminal domain alone. These findings support the model for lateral association of utrophin along the actin filament and provide the molecular basis for designing the most effective utrophin "mini-genes" for treatment of dystrophinopathies.
Rybakova
Inna N
IN
Department of Physiology, University of Wisconsin Medical School, Madison, 53706, USA.
Ervasti
James M
JM
D000199:Actins / Q000737:chemistry*; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D007700:Kinetics; D051379:Mice; D008840:Microfilament Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013049:Spectrin / Q000737:chemistry*; D013997:Time Factors; D049411:Utrophin / Q000737:chemistry*; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D000939:Epitopes; D008840:Microfilament Proteins; D011994:Recombinant Proteins; D013049:Spectrin; D049411:Utrophin; D000096985:Calponins