Binding of dystrophin's tandem calponin homology domain to F-actin is modulated by actin's structure.
Dystrophin has been shown to be associated in cells with actin bundles. Dys-246, an N-terminal recombinant protein encoding the first 246 residues of dystrophin, includes two calponin-homology (CH) domains, and is similar to a large class of F-actin cross-linking proteins including alpha-actinin, fimbrin, and spectrin. It has been shown that expression or microinjection of amino-terminal fragments of dystrophin or the closely related utrophin resulted in the localization of these protein domains to actin bundles. However, in vitro studies have failed to detect any bundling of actin by either intact dystrophin or Dys-246. We show here that the structure of F-actin can be modulated so that there are two modes of Dys-246 binding, from bundling actin filaments to only binding to single filaments. The changes in F-actin structure that allow Dys-246 to bundle filaments are induced by covalent modification of Cys-374, proteolytic cleavage of F-actin's C-terminus, mutation of yeast actin's N-terminus, and different buffers. The present results suggest that F-actin's structural state can have a large influence on the nature of actin's interaction with other proteins, and these different states need to be considered when conducting in vitro assays.
Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
A
A
Orlova
I N
IN
Rybakova
E
E
Prochniewicz
D D
DD
Thomas
J M
JM
Ervasti
E H
EH
Egelman
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D016189:Dystrophin / Q000737:chemistry*; D004923:Erythrosine / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006531:HEPES / Q000378:metabolism; D008840:Microfilament Proteins; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014325:Tromethamine / Q000378:metabolism; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D016189:Dystrophin; D004923:Erythrosine; D005456:Fluorescent Dyes; D006531:HEPES; D008840:Microfilament Proteins; D014325:Tromethamine; D000096985:Calponins