title: Functional and structural relationship between the calmodulin-binding, actin-binding, and actomyosin-ATPase inhibitory domains on the C terminus of smooth muscle caldesmon.
abstract: 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.
authors.affiliation: Department of Pathobiology and the Division of Urology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Yang
authors.forename: S
authors.initials: S
authors.lastname: Chacko
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002645:Chickens; D005895:Gizzard, Avian / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000037:antagonists & inhibitors*; D010449:Peptide Mapping; D011485:Protein Binding; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
chemical_list: D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D009218:Myosins