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 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 calmodulin-binding determinant, and the domain that is responsible for the inhibition of the actin-activated ATPase of myosin.
Wang
Z
Z
Department of Pathobiology, University of Pennsylvania, Philadelphia 19104, USA.
Horiuchi
K Y
KY
Chacko
S
S
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002645:Chickens; D017931:DNA Primers / Q000737:chemistry; D008840:Microfilament Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009218:Myosins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins; D017384:Sequence Deletion; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D008840:Microfilament Proteins; D011994:Recombinant Proteins; D014335:Tropomyosin; D009218:Myosins