Kinetic analysis of the calmodulin-binding region of the plasma membrane calcium pump isoform 4b.
The sequence L(1086)RRGQILWFRGLNRIQTQIKVVKAFHSS(1113) (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 (C28NDelta3, C28NDelta5, or C28NDelta6) 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 C28NDelta6 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 I(1091)LWFRGLNRIQTQIKVVKAF(1110) (C25NDelta5) is required to reproduce the calmodulin-binding properties of C28. When F(1110) was replaced by A, the TA-calmodulin association and dissociation kinetics resembled C23 kinetics, but changing V(1107) to A produced a smaller effect, suggesting that F(1110), rather than V(1107), is the main anchor for the N-terminal lobe of calmodulin in PMCA4b.
Penheiter
Alan R
AR
Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55901, USA.
Filoteo
Adelaida G
AG
Penniston
John T
JT
Caride
Ariel J
AJ
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D014227:Triazines / Q000378:metabolism
C404210:2-chloro-(6-(4-N,N-diethylaminophenyl)-1,4,5-triazin-4-yl)calmodulin; D002147:Calmodulin; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D010446:Peptide Fragments; D014227:Triazines; C507517:ATP2B4 protein, human; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases