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

• Published in: Biochemistry (Easton) 2007-04, Vol.46 (13), p.4045-4054
• Main Authors: Slaughter, Brian D, Urbauer, Ramona J Bieber, Urbauer, Jeffrey L, Johnson, Carey K
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Calmodulin - metabolism; Cell Membrane - enzymology; Entropy; Fluorescent Dyes - chemistry; Kinetics; Maleimides - chemistry; Methionine - chemistry; Models, Chemical; Oxidation-Reduction; Plasma Membrane Calcium-Transporting ATPases - metabolism; Protein Structure, Tertiary
• ISSN: 0006-2960
• DOI: 10.1021/bi602481u

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.