Amino acids Lys-Asp-Asp-Lys-Pro-Val402 in the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum are critical for functional association with phospholamban.
Phospholamban interacts with SERCA2 (sarco/endoplasmic reticulum calcium ATPase isoform 2) but not SERCA3. The use of chimeric SERCA2/SERCA3 molecules has revealed that amino acids 336-412 in the phosphorylation domain of SERCA2 are essential for functional association with phospholamban (Toyofuku, T., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1993) J. Biol. Chem. 268, 2809-2815). When mutations were made in SERCA2 between amino acids 336 and 412 and the mutants were coexpressed with phospholamban, only mutation of amino acids Lys397 to Val402 affected phospholamban association with the Ca(2+)-ATPase. A chimeric Ca(2+)-ATPase, CH2, was created in which the phosphorylation domain of SERCA2 was replaced with that of SERCA3, disrupting functional interaction with phospholamban. The SERCA3 sequence QGEQLV402 was then mutated to the corresponding SERCA2 sequence, KDD-KPV402, and to the sequence KGEKPV402, resulting in restoration of functional interaction with phospholamban. Mutation to KGNKPV402 or to QGEQPV402 did not restore functional interaction with phospholamban. These results demonstrate that amino acids Lys397-Val402 comprise the interaction site with phospholamban in SERCA2, and probably also in SERCA1, and that the appropriate balance of charged and hydrophobic residues is an important feature of the interaction.
Toyofuku
T
T
Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Ontario, Canada.
Kurzydlowski
K
K
Tada
M
M
MacLennan
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000099211:Phospholamban