Abstract
We previously identified a cluster of basic spectrin-like repeats in the dystrophin rod domain that binds F-actin through electrostatic interactions (Amann, K. J., Renley, B. A., and Ervasti, J. M. (1998) J. Biol. Chem. 273, 28419-28423). Because of the importance of actin binding to the presumed physiological role of dystrophin, we sought to determine whether the autosomal homologue of dystrophin, utrophin, shared this rod domain actin binding activity. We therefore produced recombinant proteins representing the cluster of basic repeats of the dystrophin rod domain (DYSR11-17) or the homologous region of the utrophin rod domain (UTROR11-16). Although UTROR11-16 is 64% similar and 41% identical to DYSR11-17, UTROR11-16 (pI = 4. 86) lacks the basic character of the repeats found in DYSR11-17 (pI = 7.44). By circular dichroism, gel filtration, and sedimentation velocity analysis, we determined that each purified recombinant protein had adopted a stable, predominantly alpha-helical fold and existed as a highly soluble monomer. DYSR11-17 bound F-actin with an apparent K(d) of 7.3 +/- 1.3 microM and a molar stoichiometry of 1:5. Significantly, UTROR11-16 failed to bind F-actin at concentrations as high as 100 microM. We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin.
Highlights
Dystrophin is the 427-kDa protein product of the Duchenne muscular dystrophy locus
We had previously proposed a mechanism for the interaction of the dystrophin rod domain with F-actin, whereby a cluster of basic spectrin-like repeats near the middle of the rod domain interacts with the actin filament via electrostatic interactions [1]
Sequence analysis further indicated that three of the four spectrin-like repeats in DYS1416 were basic, while only one of four repeats in DYS1030 was basic, which led us to hypothesize that DYS1416 bound F-actin largely through electrostatic interactions
Summary
Vol 274, No 50, Issue of December 10, pp. 35375–35380, 1999 Printed in U.S.A. From the ‡Graduate Program in Cellular and Molecular Biology and ¶Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706 and the §Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, Minnesota 55455. UTROR11–16 failed to bind F-actin at concentrations as high as 100 M We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin. To determine whether dystrophin rod domain repeats 11–17 form a more extensive lateral association than do repeats 11–14 and to determine whether utrophin’s central rod domain is capable of binding F-actin, we produced recombinant proteins representing the complete cluster of basic repeats of the dystrophin rod (DYSR11–17) or the corresponding region from utrophin (UTROR11–16). The failure of the homologous, but acidic, utrophin middle rod domain to bind actin is further support for the electrostatic nature of the interaction of the basic dystrophin repeats with actin and suggests that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin
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