Identification of the functionally relevant calmodulin binding site in smooth muscle caldesmon.
The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-666 (Zhan, Q., Wong, S. S., and Wang, C.-L. A. (1991) J. Biol. Chem. 266, 21810-21814). Recently, another segment (site B), Asn-675 to Lys-695, was reported to bind CaM (Mezgueldi, M., Derancourt, J., Calas, B., Kassab, R., and Fattoum, A. (1994) J. Biol. Chem. 269, 12824-12832). To assess the functional relevance of these two putative CaM-binding sites, we have examined three synthetic peptides regarding their effects on CaM's ability to reverse CaD-induced inhibition of actomyosin ATPase activity: GS17C (Gly-651 to Ser-667), VG29C (Val-685 to Gly-713), each containing one CaM-binding site, and MG56C (Met-658 to Gly-713), which contains both sites. We found that although VG29C did bind CaM, its affinity was weakened by GS17C, and it failed to compete with CaD for CaM under the conditions where GS17C effectively displaced CaD from CaM. MG56C had an effect similar to that of GS17C. These experiments demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.
Zhuang
S
S
Muscle Research Group, Boston Biomedical Research Institute, Massachusetts 02114, USA.
Wang
E
E
Wang
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D009218:Myosins