THE 4TH EF-HAND OF CALMODULIN AND ITS HELIX-LOOP-HELIX COMPONENTS - IMPACT ON CALCIUM-BINDING AND ENZYME ACTIVATION

CaM (4 cTnC) is a calmodulin-cardiac troponin C chimeric protein conta ining the first, second, and third calcium-binding EF-hands of calmodu lin (CaM) and the fourth EF-hand of cardiac troponin C (cTnC) [George, S. E., Su, Z., Fan, D., & Means, A. R. (1993) J. Biol. Chem. 268, 252 13-25220]. CaM (4 cTnC) showed 2-fold-enhanced carboxy-terminal Ca2+ a ffinity relative to CaM and also exhibited impaired activation of the CaM-regulated enzymes smooth muscle myosin light chain kinase (smMLCK) , neuronal nitric oxide synthase (nNOS): and phosphodiesterase (PDE). To investigate the molecular basis for these effects, we constructed ( 1) additional chimeras, replacing most of CaM helix 7, Ca2+-binding lo op 4, and helix 8 with the corresponding helices and loops of cTnC; an d (2) point mutants in the fourth EF-hand of CaM. Replacement of CaM's fourth loop with the corresponding loop of cTnC enhanced Ca2+ affinit y by over 3-fold through an increase in the Ca2+ on rate and also redu ced cooperativity of Ca2+ binding. In contrast, substitution of CaM he lix 7 or 8 modestly decreased Ca2+ affinity by increasing the Ca2+ off rate, without impairment of cooperativity. All three of the helix and loop chimeras fully activate smMLCK and nNOS, whereas the other two c himeras retained about 80% of the maximal smMLCK and nNOS activation o bserved with CaM.