Characterization of the Functional Domains on the C-terminal Region of Caldesmon Using Full-length and Mutant Caldesmon Molecules

Abstract

A series of C-terminal deletion mutants of chicken gizzard smooth muscle caldesmon (CaD) were made using a polymerase chain reaction cloning strategy and a baculovirus expression system, and the precise locations of the functional domains of CaD involved in the regulation of actomyosin ATPase and the binding of actin, tropomyosin, and calmodulin were analyzed. Our results reveal a high affinity calmodulin-binding domain that consists of at least three calmodulin-binding determinants localized in residues 690 -717, 658 -689, and 628 -657. The residues between positions 718 and 756 and positions 598 and 627 have no detectable calmodulin-binding site. A high affinity tropomyosin-binding domain is located between residues 718 and 756. The 159 residues at the C terminus of CaD contain multiple actin-binding determinants; the major ones are localized in the regions between residues 718 and 756 and residues 690 and 717. The amino acid residues between positions 718 and 756 contain the major determinant involved in the inhibition of the actin activation of smooth muscle myosin ATPase since CaD-(1-717) caused only 30% of the inhibition produced by the full-length CaD. Further deletion between residues 690 and 717 (CaD-(1-689)) revealed a low level (10% of that seen for full-length CaD) of inhibition of the actomyosin ATPase. These data clearly demonstrate that the region of the last 66 amino acid residues at the CaD C terminus contains two or more major actin-binding motifs, one tropomyosin-binding domain, one high affinity calmodulinbinding determinant, and the domain that is responsible for the inhibition of the actin-activated ATPase of myosin.The thin filament-associated protein caldesmon (CaD) 1 binds to actin, calmodulin, and tropomyosin, and the binding of this protein to actin or tropomyosin-actin inhibits the actin-activated Mg-ATPase activity of phosphorylated smooth muscle myosin (1-5). This inhibition is released by the binding of calmodulin to caldesmon in the presence of Ca 2ϩ (1-3, 6, 7). Furthermore, tropomyosin amplifies the caldesmon-induced inhibition of actin-activated myosin ATPase activity (1,3,6). CaD is thought to be a protein involved in the thin filament-mediated regulation that complements the myosin-mediated regulation of smooth muscle myosin via myosin light chain phosphorylation and dephosphorylation (Refs. 8 -10; for review, see Ref. 11).Structurally, CaD is a long asymmetric monomeric (80 ϫ 2 nm) molecule that is localized along the actin filament, covering 7-14 actin monomers (12)(13)(14). While the CaD C terminus binds to actin, the N-terminal region of CaD binds to the S-2 regions of the myosin (15, 16). An understanding of the structural and functional domains on the CaD molecule, responsible for its interaction with myosin, actin, calmodulin, and tropomyosin, is essential to elucidate the mechanism by which CaD down-regulates actomyosin ATPase activity. Previous studies (17-19) have demonstrated an actin-and a calmodulin-binding region on the C-terminal proteolytic fragment of ϳ38 kDa...

Tryptophan Residues in Caldesmon Are Major Determinants for Calmodulin Binding

“…Site B′, in contrast, may act as a secondary site whose binding is easily reversible depending on the environment surrounding the thin filament. The weak interaction between site B′ and calmodulin may also explain why some have observed an interaction between calmodulin and caldesmon fragments containing site B′ (Huber et al, 1996;Marston et al, 1994), while others (Wang et al, 1996) did not detect significant changes in binding when the region containing site B′ (residues 718-756) was truncated from caldesmon.…”

“…Recent NMR data by Huber et al (1996) showed the distinct Ca 2+ -calmodulin interaction with Trp residues 692 and 722 at sites B and B′, respectively. Another report by Wang et al (1996) has shown that there are three calmodulin-binding determinants localized within residues 628-717, which encompasses only sites A and B. However, deletion of residues 718-756, containing site B′, had no effect on calmodulin binding.…”

“…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).…”

Caldesmon and the Regulation of Cytoskeletal Functions

“…65 Subsequently, it was determined that the Tm-binding sites were localized in the C-terminal region. [66][67][68][69][70] This region of the last ~300 amino acid residues not only contains the actin-and the CaM-binding sites, but also harbors TnT-analogous sequences that interact with Tm. 71 Apparently there are several peptide segments in this region that bind actin, Tm and CaM independently (see, for example, 72).…”

“…83 Indeed, structural studies showed the C-terminal region of CaD contains multiple β-turns, which allows for plenty of flexibility and facilitates docking on actin filaments. 84 Functionally, the C-terminal region of CaD harbors actin-, CaM-and Tm-binding sites, 68,[85][86][87][88][89] whereas the N-terminal domain contains the major myosin-binding sites [90][91][92] and also interacts with CaM and actin. 93,94 The actin-and myosin-binding capacity enables CaD to play a role in bringing the thin filaments close to the thick filaments (see below).…”