Abstract
We have investigated the mechanisms of activation of the plasma membrane (PM) Ca-ATPase by calmodulin (CaM), which result in enhanced calcium transport rates and the maintenance of low intracellular calcium levels. We have isolated the amino- or carboxyl-terminal domains of CaM (i.e. CaMN or CaMC), permitting an identification of their relative specificity for binding to sites on either the PM Ca-ATPase or a peptide (C28W) corresponding to the CaM-binding sequence. We find that either CaMN or CaMC alone is capable of productive interactions with the PM Ca-ATPase that induces enzyme activation. There are, however, large differences in the affinity and specificity of binding between CaMN and CaMC and either C28W or the PM Ca-ATPase. The initial binding interaction between CaMC and the PM Ca-ATPase is highly specific, having approximately 10,000-fold greater affinity in comparison with CaMN. However, following the initial association of either CaMC or CaMN, there is a 300-fold enhancement in the affinity of CaMN for the secondary binding site. Thus, while CaMC binds with a high affinity to the two CaM-binding sites within the PM Ca-ATPase in a sequential manner, CaMN binds cooperatively with a lower affinity to both binding sites. These large differences in the binding affinities and specificities of the amino- and carboxyl-terminal domains ensure that CaM binding to the PM Ca-ATPase normally involves the formation of a specific complex in which the initial high affinity association of the carboxyl-terminal domain promotes the association of the amino-terminal domain necessary for enzyme activation.
Highlights
A range of diverse metabolic activities involved in intracellular signaling is mediated by calmodulin (CaM),1 which functions as the major calcium sensor in all eukaryotes
It is possible to isolate an amino-terminal fragment containing amino acids Ala1–Lys75 (CaMNЈ) and Asp78 -Lys148 (CaMC) using weak anion exchange HPLC. The identity of these fragments was determined using electrospray ionization mass spectrometry; the observed average molecular masses of CaMNЈ and corresponding to amino acids –148 (CaMC) are 8316 Ϯ 1 and 8147 Ϯ 1 Da, respectively, in close agreement with expected monoisotopic molecular masses of 8316.2 and 8147.8 Da for these CaM fragments. These results suggest that previous measurements in which the amino-terminal domain of CaM was reported to be unable to activate the plasma membrane (PM) Ca-ATPase probably involved CaMNЈ [16]
In case Met76 and Lys77 located in the linker region between the amino- and carboxyl-terminal domains might play a role in facilitating target protein binding, we have cloned and expressed the amino-terminal domain of CaM containing amino acids Ala1–Lys77 (CaMN) in E. coli
Summary
A range of diverse metabolic activities involved in intracellular signaling is mediated by calmodulin (CaM), which functions as the major calcium sensor in all eukaryotes. Differences in the interactions between the amino- and carboxyl-terminal domains of CaM and their corresponding binding sites within target proteins have been suggested to play essential roles in target protein activation In this respect, previous measurements have demonstrated that the binding preferences of individual CaM-binding domains for sites within the CaM-binding sequence of skeletal myosin light chain kinase are relatively small (i.e. less than 1 kcal/mol), suggesting the possibility of multiple conformations of bound CaM that could function to regulate the extent of target protein activation observed in the presence of saturating CaM concentrations [9, 15]. The substantially higher affinity of the carboxyl-terminal domain relative to the aminoterminal domain suggests that activation of the PM Ca-ATPase normally involves the initial association between the carboxylterminal domain of CaM to a single site within the CaMbinding sequence of the PM Ca-ATPase, followed by subsequent association with the amino-terminal domain
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