IDENTIFICATION OF THE FUNCTIONALLY RELEVANT CALMODULIN-BINDING SITE IN SMOOTH-MUSCLE CALDESMON

The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosi n ATPase activity. me have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Se r-666 (Zhan, Q., Wong, S. S., and Wang, C.-L. A. (1991) J. Biol. Chem. 266, 21810-21814). Recently, another segment (site B), Asn-675 to Lys -695, was reported to bind CaM (Mezgueldi, M., Derancourt, J., Calas, B., Kassab, R., and Fattoum, A (1994) J. Biol. Chem. 269, 12824-12832) . To assess the functional relevance of these two putative CaM-binding sites, we have examined three synthetic peptides regarding their effe cts on CaM's ability to reverse CaD-induced inhibition of actomyosin A TPase activity: GS17C (Gly-651 to Ser-667), VG29C (Val-685 to Gly-713) , each containing one CaM-binding site, and MG56C (Met-658 to Gly-713) , which contains both sites. We found that although VG29C did bind CaM , its affinity was weakened by GS17C, and it failed to compete with Ca D for CaM under the conditions where GS17C effectively displaced CaD f rom CaM. MG56C had an effect similar to that of GS17C. These experimen ts demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.