Interaction of smooth muscle caldesmon with calmodulin mutants.
The interaction of avian smooth muscle caldesmon with calmodulin (CaM) was investigated by studying the ability of selected mutant calmodulins to induce fluorescence changes in caldesmon. Different types of CaM mutants were used including point charge mutants, cluster mutations, and mutations which alter the calcium binding of CaM. The caldesmon binding properties were only slightly affected by E84K-CaM or by the double mutation E84Q/E120Q-CaM. Affinity of calmodulin to caldesmon was decreased 2-4 times by point mutation G33V-CaM, double mutation E84K/E120K-CaM, deletion of residues 82-84, and by cluster mutations DEE118-120-->KKK or EEE82-84-->KKK. Mutations of the first (E31A-CaM) and the second (E67A-CaM) calcium binding sites reduced the affinity of calmodulin to caldesmon by at least 5-fold; in addition these calmodulin mutants exhibited smaller changes in the fluorescence spectra of caldesmon. Simultaneous mutation of the two negatively charged clusters of calmodulin EEE82-84-->KKK and DEE118-120-->KKK resulted in a more than 15-fold decrease in the affinity of calmodulin for caldesmon. The data indicate that charged and uncharged amino acids in both halves of CaM play an important role in the binding of calmodulin to caldesmon, and that Ca2+ binding must be maintained in the amino-terminal sites for maximal interaction with caldesmon.
Medvedeva
M V
MV
Department of Biochemistry, School of Biology, Moscow State University, Russian Federation.
Bushueva
T L
TL
Shirinsky
V P
VP
Lukas
T J
TJ
Watterson
D M
DM
Gusev
N B
NB
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D004372:Ducks; D009130:Muscle, Smooth / Q000378:metabolism*; D009154:Mutation; D010764:Phosphorylase Kinase / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010764:Phosphorylase Kinase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases