Functional and structural relationship between the calmodulin-binding, actin-binding, and actomyosin-ATPase inhibitory domains on the C terminus of smooth muscle caldesmon.

Z Wang, Z Q Yang, S Chacko

The Journal of biological chemistry 1997 Jul 04

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Multiple functional domains responsible for calmodulin (CaM) binding and actin-binding/actomyosin ATPase inhibition are present in the region between residues 598-756 of the chicken gizzard smooth muscle caldesmon (CaD) molecule. To precisely localize these functional domains and to further elucidate the structural basis of these domains, we analyzed a series of purified mutants of chicken gizzard smooth muscle CaD generated by internal deletions of amino acid sequences and expression in a baculovirus expression system. Our results demonstrate that, in addition to a strong actin-binding site sequence between residues 718-723 (Wang, Z., and Chacko, S. (1996) J. Biol. Chem. 271, 25707-25714), two weak actin-binding motifs are present in the regions between residues 690-699 and 650-666. These weak actin-binding regions function independently and are associated with weak actomyosin inhibitory activity. Analysis of the CaM-binding sites A (residues 658-666) and B (residues 690-695), the major CaM-binding sites in the C-terminal region of CaD, provided direct evidence for the involvement of both CaM-binding sites in the CaM-mediated reversal of the inhibition of actomyosin ATPase activity by CaD and for the functional independence of the two CaM-binding sites. Furthermore, the sequences between residues 598-649, upstream of CaM-binding site A, and 700-717, downstream of CaM-binding site B, appear to have no effect on either actin-binding or CaM-binding. The data also suggest that both CaM-binding sites A and B structurally overlap or lie in close proximity to the adjacent weak actin-binding sites and weak actomyosin ATPase inhibitory determinants.

Citation

Z Wang, Z Q Yang, S Chacko. Functional and structural relationship between the calmodulin-binding, actin-binding, and actomyosin-ATPase inhibitory domains on the C terminus of smooth muscle caldesmon. The Journal of biological chemistry. 1997 Jul 04;272(27):16896-903