Calmodulin-cardiac troponin C chimeras. Effects of domain exchange on calcium binding and enzyme activation.
Calmodulin (CaM) and the cardiac isoform of troponin C (cTnC) are close structural homologs, but cTnC cannot activate most CaM target enzymes. To investigate structure-function relationships, we constructed a series of CaM.cTnC chimeras and determined their ability to bind Ca2+ and activate CaM target enzymes. Previously, we exchanged domain 1 and found that the chimeras exhibited profoundly impaired activation of smooth muscle myosin light chain kinase (smMLCK) and had differential effects on other CaM target enzymes (George, S. E., VanBerkum, M. F. A., Ono, T., Cook, R., Hanley, R. M., Putkey, J. A., and Means, A. R. (1990) J. Biol. Chem. 265, 9228-9235). One of the domain 1 chimeras was a potent competitive inhibitor of smMLCK. We now extend our study of CaM.cTnC chimeras by exchanging all of the remaining functional domains of CaM and cTnC. We determined the ability of the chimeras to bind Ca2+ and activate phosphodiesterase (PDE) and smMLCK. Chimeras containing both domains 3 and 4 of cTnC exhibited high affinity Ca2+ binding that was indistinguishable from cTnC, whereas chimeras containing either domain 3 or 4 of cTnC demonstrated Ca2+ affinity that was intermediate between CaM and cTnC. All of the CaM.cTnC chimeras showed near-maximal PDE activation but required 5-775-fold higher concentrations than CaM to produce half-maximal PDE activation. In contrast, all of the chimeras showed impaired ability to activate smMLCK, and some were potent competitive inhibitors of smMLCK activation by CaM.
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Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D014336:Troponin / Q000235:genetics*; D019209:Troponin C
D002147:Calmodulin; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014336:Troponin; D019209:Troponin C; D009219:Myosin-Light-Chain Kinase; D010727:Phosphoric Diester Hydrolases; D002118:Calcium