The Fourth EF-Hand of Calmodulin and Its Helix−Loop−Helix Components:  Impact on Calcium Binding and Enzyme Activation

Abstract

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 activated PDE, with minor shifts in Kact. CaM (helix 7 cTnC) showed a significantly impaired ability to activate smMLCK and nNOS, whereas the other two chimeras retained about 80% of the maximal smMLCK and nNOS activation observed with CaM.

Neuronal Nitric-oxide Synthase Interaction with Calmodulin-Troponin C Chimeras

“…12) to determine if an attached oxygenase domain would affect response to each chimera. In all cases, the chimeras were present during assay at concentrations previously shown to saturate nNOS (8).…”

“…The recombinant nNOS reductase domain (amino acids 724 -1429) containing the CaM binding site was expressed in the yeast Pichia pastoris and purified as described previously (12). The CaM-TnC chimeras were expressed in Escherichia coli and purified as described previously (8). All other reagents and materials were obtained from Sigma or from sources reported previously (12,14).…”

“…6). CaM may be unique among the Ca 2ϩ -binding proteins in activating NO synthesis (7,8), and the mechanism is of current interest. Our studies with nNOS show that CaM controls electron transfer at two points in the enzyme (Fig.…”

“…Despite this structural similarity, TnC can neither bind to nor activate nNOS (7). Because of the divergence between structure and function, CaM-TnC chimeras are informative probes of the CaM-nNOS interaction (7,8). For example, several CaM-TnC chimeras bound avidly to nNOS, yet failed to activate NO synthesis by the enzyme, whereas others both bound and activated.…”

“…This demonstrated that enzyme binding and stimulation of NO synthesis are distinct and separable. The initial studies further established that the domain 1-domain 3 "latch" region of CaM played a critical role in inducing NO production (8).…”