Identification of the Functionally Relevant Calmodulin Binding Site in Smooth Muscle Caldesmon

Abstract

The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-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 effects on CaM's ability to reverse CaD-induced inhibition of actomyosin ATPase 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 CaD for CaM under the conditions where GS17C effectively displaced CaD from CaM. MG56C had an effect similar to that of GS17C. These experiments demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.

Heat Treatment Could Affect the Biochemical Properties of Caldesmon

“…Smooth muscle CaD (Lynch and Bretscher, 1986;Wang, 1988), myosin (Ikebe et al, 1978), MLCK (Walsh et al, 1983), and tropomyosin (Graceffa, 1987) were purified from chicken gizzard. Purified smooth muscle myosin was phosphorylated by MLCK as described previously (Zhuang et al, 1995).…”

Tryptophan Residues in Caldesmon Are Major Determinants for Calmodulin Binding

“…Smooth muscle h-caldesmon has a M r of 87 000 and contains three structurally and functionally distinct domains: an NH 2 -terminal domain (residues 1-250), a middle domain (residues 251-400), and a COOH-terminal domain (residues 401-756). While all three domains have been shown to bind tropomyosin in Vitro (Redwood & Marston, 1993;Graceffa, 1987;Watson et al, 1990), the NH 2 -terminal domain also binds myosin (Velaz et al, 1990;Ikebe & Reardon, 1988), and the COOHterminal domain interacts with actin (Bartegi et al, 1990;Mezgueldi et al, 1994) and the Ca 2+ -binding proteins calmodulin (Zhan et al, 1991;Zhuang et al, 1995;Marston et al, 1994) and caltropin (Mani et al, 1992). Both NH 2 -and COOH-terminal domains of smooth muscle caldesmon are conserved in nonmuscle caldesmon with similar primary structures and functions (Hayashi et al, 1991;Bryan & Lee, 1991).…”

“…It is generally agreed that there are multiple Ca 2+ -calmodulin-binding sites at the COOHterminal domain of caldesmon. However, there is disagreement concerning the location of the sites and the relative contribution of each site in binding to calmodulin and in the release of caldesmon inhibition of actomyosin ATPase (Wang et al, 1996;Marston et al, 1994;Zhuang et al, 1995;Huber et al, 1996).…”

Functional and Structural Relationship between the Calmodulin-binding, Actin-binding, and Actomyosin-ATPase Inhibitory Domains on the C Terminus of Smooth Muscle Caldesmon

“…The data from Marston et al (34) favor CaM-binding site B, but not CaM-binding site A, as the function-associated CaM-binding site because a synthetic peptide corresponding to the sequence Ser 657 -Gly 670 did not release the CaM-induced reversal of the inhibition of actomyosin ATPase activity by CaD. By contrast, Zhuang et al (47), using a similar research approach, concluded that CaM-binding site A, but not CaM-binding site B, is involved in the CaM-CaD interaction for reversal of the CaD-induced inhibition. One reason for this discrepancy may be the lack of amino acids adjacent to CaM binding sites A or B needed for the protein conformation required for the proteinprotein interaction in these studies.…”

“…While the major CaM-binding sites in CaD molecule are well established, there is controversy about the functional significance of CaM-binding sites A and B in CaM-induced reversal for inhibition of actomyosin ATPase activity by CaD (34,47). The data from Marston et al (34) favor CaM-binding site B, but not CaM-binding site A, as the function-associated CaM-binding site because a synthetic peptide corresponding to the sequence Ser 657 -Gly 670 did not release the CaM-induced reversal of the inhibition of actomyosin ATPase activity by CaD.…”