CHARACTERIZATION OF THE FUNCTIONAL DOMAINS ON THE C-TERMINAL REGION OF CALDESMON USING FULL-LENGTH AND MUTANT CALDESMON MOLECULES

A series of C-terminal deletion mutants of chicken gizzard smooth musc le caldesmon (CaD) were made using a polymerase chain reaction cloning strategy and a baculovirus expression system, and the precise locatio ns of the functional domains of CaD involved in the regulation of acto myosin ATPase and the binding of actin, tropomyosin, and calmodulin we re analyzed. Our results reveal a high affinity calmodulin-binding dom ain that consists of at least three calmodulin-binding determinants lo calized in residues 690-717, 658-689, and 628-657. The residues betwee n positions 718 and 756 and positions 598 and 627 have no detectable c almodulin-binding site. A high affinity tropomyosin-binding domain is located between residues 718 and 756. The 159 residues at the C termin us 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 co ntain the major determinant involved in the inhibition of the actin ac tivation of smooth muscle myosin ATPase since CaD-(1-717) caused only 30% of the inhibition produced by the full-length CaD. Further deletio n 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 ATP ase. These data clearly demonstrate that the region of the last 66 ami no acid residues at the CaD C terminus contains two or more major acti n-binding motifs, one tropomyosin-binding domain, one high affinity ca lmodulin-binding determinant, and the domain that is responsible for t he inhibition of the actin-activated ATPase of myosin.