Mutagenesis analysis of functionally important domains within the C-terminal end of smooth muscle caldesmon.
The ability of chicken gizzard smooth muscle caldesmon (CaD) to inhibit actomyosin ATPase activity is due mainly to an inhibitory domain that resides within the C-terminal 67 amino acid residues of the CaD molecule. In the present study, a series of C-terminal truncation and internal deletion mutants of chicken gizzard smooth muscle CaD were systematically designed using a site-directed mutagenesis approach, and these mutant proteins were overexpressed in a baculovirus expression system. Analysis of actin binding and inhibition of actomyosin ATPase activity using these mutants identified a strong actin-binding motif of 6 amino acid residues (from Lys718 to Glu723), which also form the core sequence for CaD-induced inhibition of actomyosin ATPase. However, maximal inhibition by CaD requires the presence of residues 728-731, which are not associated with actin binding. Our data provide direct evidence for the requirement of actin binding to a specific region in CaD for CaD-induced inhibition of actin activation of smooth muscle myosin ATPase. Furthermore, our findings also show that the region between residues 690 and 717 is responsible for the weak inhibition of actomyosin ATPase and reveal that the inhibitory determinants located in the regions between residues 690 and 717 and residues 718 and 756 can function independently.
Wang
Z
Z
Department of Pathobiology, and the Division of Urology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Chacko
S
S
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002645:Chickens; D017931:DNA Primers; D005895:Gizzard, Avian; D018698:Glutamic Acid; D007700:Kinetics; D008239:Lysine; D009130:Muscle, Smooth / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D017384:Sequence Deletion; D014162:Transfection
D000199:Actins; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D018698:Glutamic Acid; D009218:Myosins; D008239:Lysine