Localization of Protein Regions Involved in the Interaction between Calponin and Myosin

doi:10.1074/jbc.272.17.11142

Localization of Protein Regions Involved in the Interaction between Calponin and Myosin*

From the^‡ Muscle Research Group, Boston Biomedical Research Institute, Boston, Massachusetts 02114, the
^¶ Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, and the
^∥ Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

^§ To whom correspondence should be addressed:
Harvard University, West Roxbury VA Medical Center, 1400 VFW Pkwy., Research 151, Bldg. 3, Rm. 2B102, West Roxbury, MA 02132.
Tel: 617-323-7700 ext. 6195; Fax: 617-363-5592; E-mail: tao{at}bbri.harvard.edu

Abstract

Calponin is a 33-kDa smooth muscle-specific protein that has been suggested to play a role in muscle contractility. It has previously been shown to interact with actin, tropomyosin, and calmodulin. More recently we showed that calponin also interacts with myosin (Szymanski, P. T., and Tao, T. (1993) FEBS Lett. 331, 256-259). In the present study we used a combination of co-sedimentation and fluorescence assays to localize the regions in myosin and calponin that are involved in the interaction between these two proteins. We found that recombinant chicken gizzard α-calponin co-sediments with myosin rod and, to a lesser extent, with light meromyosin. Fluorescently labeled recombinant calponin shows interaction with heavy meromyosin and myosin subfragment 2 but not subfragment 1. A fragment comprising residues 7-182 and a synthetic peptide spanning residues 146-176 of calponin co-sediment with myosin, but fragments comprising residues 7-144 and 183-292 do not. Our results indicate that there are calponin binding sites in the subfragment 2 and light meromyosin regions of myosin, and that the region comprising residues 145-182 of calponin mediates its interaction with myosin.

Footnotes

↵* This work was supported by National Institutes of Health Grant PO1-AR41637 (to T. T.) and American Heart Association Grant 13-523-934 (to P. T. S.). 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.
↵¹ The abbreviations used are:

CaP

calponin

Rα CaP

recombinant α-isoform of chicken gizzard calponin

HMM

heavy meromyosin

LMM

light meromyosin

S1

myosin subfragment 1

S2

myosin subfragment 2

DAN-Rα CaP

N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine-labeled Rα CaP

PAGE

polyacrylamide gel electrophoresis

Hepes

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
↵² P. T. Szymanski and T. Tao, unpublished observations.
- Received October 1, 1996.
- Revision received January 22, 1997.