title: Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca(2+)-ATPase: kinetic pathway and effects of methionine oxidation.
abstract: Calmodulin (CaM) binds to a domain near the C-terminus of the plasma membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide [C28W(1b)] corresponding to the CaM-binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism in which an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the poorer effectiveness of oxidized CaM in activating the Ca2+ pump.
authors.affiliation: Department of Chemistry, 1251 Wescoe Hall Drive, University of Kansas, Lawrence, Kansas 66045-7582, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Slaughter
authors.forename: Ramona J Bieber
authors.initials: RJ
authors.lastname: Urbauer
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
authors.forename: Carey K
authors.initials: CK
authors.lastname: Johnson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D019277:Entropy; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry; D008715:Methionine / Q000737:chemistry*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: C504424:Alexa Fluor 488 C5-maleimide; D002147:Calmodulin; D005456:Fluorescent Dyes; D008301:Maleimides; D008715:Methionine; D053499:Plasma Membrane Calcium-Transporting ATPases