Strong Interaction between Caldesmon and Calponin*
- ‡ To whom correspondence should be addressed: Boston Biomedical Research Institute, 20 Staniford St., Boston, MA 02114. Tel.: 617-742-2010 (ext. 367); Fax: 617-523-6649; E-mail: graceffa{at}bbri.harvard.edu.
Abstract
Caldesmon was labeled at either Cys-153 in the NH2-terminal domain or Cys-580 in the COOH-terminal domain with a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan) fluorescence probe. The addition of smooth muscle calponin to Cys-580-labeled caldesmon resulted in an 18% drop in fluorescence intensity, which titrated with a stoichiometry of 0.9 and a binding constant of 9.5 × 107 M−1. For Cys-153-labeled caldesmon, there was no change in fluorescence upon adding calponin. These findings indicate strong binding between calponin and the COOH-domain of caldesmon. The association was sensitive to ionic strength, suggesting that ionic interactions between calponin, a basic protein, and caldesmon, an acidic protein, contribute to the stabilization of the protein complex. That non-muscle acidic calponin interacts with caldesmon with a much reduced association constant of 3.5 × 106 M−1 supports such a model. The binding between acidic calponin and caldesmon is strengthened to 1.8 × 107 M−1 in the presence of Ca2+, which might bind to acidic residues of the calponin and partially neutralize its negative charge. The strong, specific binding between calponin and caldesmon suggests that this interaction occurs within smooth muscle cells and possibly plays a role in the regulation of contraction.
Footnotes
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↵* This work was supported by National Institutes of Health Grants AR-30917, AR-41637, HL56035, and HL42293. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- Mops
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3-(N-morpholino)propanesulfonic acid
- MAP
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mitogen-activated protein
- acrylodan
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6-acryloyl-2-dimethylaminonaphthalene.
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↵2 W. Stafford, unpublished results.
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↵3 P. Graceffa, submitted for publication.
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- Received June 24, 1996.
- Revision received August 26, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
