Kinetic Analysis of the Calmodulin-Binding Region of the Plasma Membrane Calcium Pump Isoform 4b

The sequence L1086RRGQILWFRGLNRIQTQIKVVKAFHSS1113 (peptide C28) is responsible for calmodulin binding to PMCA4b. In this work, peptides following the above sequence were progressively shortened either at the N-terminus (C28NΔ3, C28NΔ5, or C28NΔ6) or at the C-terminus (C20, C22, C23, and C25). Competitive inhibition of PMCA activity was used to measure apparent dissociation constants of the complexes between calmodulin and C28 or progressively shortened peptides. Additionally, equilibrium titrations were used to measure the apparent dissociation constants of the various peptides with TA-calmodulin by changes in TA-calmodulin fluorescence and Trp fluorescence of the peptides. At the N-terminus, deletion of five residues did not change calmodulin affinity, but deletion of six residues resulted in a 5-fold decrease in affinity. There were no major differences in the time course of TA-CaM binding, but C28NΔ6 exhibited a different time course of Trp fluorescence change. At the C-terminus, deletion of five residues (C23) or more resulted in a net increase in fluorescence of TA-CaM upon binding, while longer peptides (C25 and C28) produced both a transient increase and a net decrease in the fluorescence of TA-CaM. Global regression analysis revealed that binding of TA-CaM to the C23 peptide could be fit by a two-step model, while longer peptides required three-step models for adequate fitting. TA-calmodulin dissociated rapidly from C23, C22, and C20, resulting in a marked increase in apparent K d. Thus, the sequence I1091LWFRGLNRIQTQIKVVKAF1110 (C25NΔ5) is required to reproduce the calmodulin-binding properties of C28. When F1110 was replaced by A, the TA-calmodulin association and dissociation kinetics resembled C23 kinetics, but changing V1107 to A produced a smaller effect, suggesting that F1110, rather than V1107, is the main anchor for the N-terminal lobe of calmodulin in PMCA4b.