The fourth 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 containing the first, second, and third calcium-binding EF-hands of calmodulin (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, 25213-25220]. CaM (4 cTnC) showed 2-fold-enhanced carboxy-terminal Ca2+ affinity 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 loop 4, and helix 8 with the corresponding helices and loops of cTnC; and (2) point mutants in the fourth EF-hand of CaM. Replacement of CaM's fourth loop with the corresponding loop of cTnC enhanced Ca2+ affinity by over 3-fold through an increase in the Ca2+ on rate and also reduced cooperativity of Ca2+ binding. In contrast, substitution of CaM helix 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 chimeras retained about 80% of the maximal smMLCK and nNOS activation observed with CaM.