title: Ordered and cooperative binding of opposing globular domains of calmodulin to the plasma membrane Ca-ATPase.
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.
authors.affiliation: Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045-2106, USA.
authors.forename: H
authors.initials: H
authors.lastname: Sun
authors.forename: T C
authors.initials: TC
authors.lastname: Squier
mesh_terms: D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D012997:Solvents; D000252:Calcium-Transporting ATPases