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 calmodulin-binding determinant, and the domain that is responsible for the inhibition of the actin-activated ATPase of myosin.
Highlights
Ated 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)
We demonstrate that the sequence that is present in the proteolytic CaD fragment of 7.3 kDa, thought to contain the region responsible for the inhibition of the actinactivated ATPase of myosin S-1 (12), is not important for the inhibition of the actin activation of smooth muscle myosin ATPase in either the presence or absence of tropomyosin
In the presence of tropomyosin under the same condition (0.06 mol of CaD bound per mol of actin), the inhibition shown by fulllength CaD and CaD-(1–717) was ϳ52 and 25%, respectively. These data suggest that tropomyosin enhances the inhibition of the actin-activated myosin ATPase activity without additional binding of CaD to actin. Our data utilizing these truncated CaD proteins reveal some important differences from the data obtained from studies using bacterially expressed or proteolytic CaD fragments, presumably due to the lack of structural association present in protein-protein interaction
Summary
Ated 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). Deletion of specific sequences from the C-terminal region, without affecting the major portion of the molecule responsible for the structural association with the actin filament, enabled us to delineate the calmodulin-, actin-, and tropomyosin-binding domains and to study the effect of these domains on the actin-activated ATPase activity of smooth muscle myosin. We demonstrate that the sequence that is present in the proteolytic CaD fragment of 7.3 kDa (residues between Leu597 and Phe665), thought to contain the region responsible for the inhibition of the actinactivated ATPase of myosin S-1 (12), is not important for the inhibition of the actin activation of smooth muscle myosin ATPase in either the presence or absence of tropomyosin. We show that deletion of a sequence (residues 718 –725) previously reported to be important for calmodulin binding to CaD, based on experiments using a bacterially expressed CaD fragment (23, 24), has no effect on calmodulin binding
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