The calmodulin-binding site of the plasma membrane Ca2+ pump interacts with the transduction domain of the enzyme.
Calpain proteolysis of the plasma membrane Ca2+ pump removes a C-terminal 14-kDa portion which includes the calmodulin-binding domain. This produces a fully activated 124-kDa fragment, which can be inhibited by synthetic versions of the calmodulin-binding domain. The inhibition is strongest when Trp-8 in the latter domain is replaced by a Tyr residue (Falchetto, R., Vorherr, T., Brunner, J., & Carafoli, E., 1991, J. Biol. Chem. 266, 2930-2936). In the present study, the N-terminus of the 28-residue synthetic calmodulin-binding domain was acetylated with 3H-acetic anhydride, and Phe in position 25 was replaced by a phenylalanine derivatized with a diazirine-based, photoactivatable carbene precursor. This peptide (C28WC*) inhibited the fully active 124-kDa fragment of the pump and became cross-linked to it upon photolysis. After proteolysis of the fragment with Asp-N or Staphylococcus aureus V8 (Glu-C) protease, labeled peptides were isolated by reversed-phase high-performance liquid chromatography and subjected to Edman sequence analysis. The peptides originated from a region of the pump located within the unit protruding into the cytoplasm between transmembrane domain two and three. This unit has been proposed to be the site of the energy transduction domain, which would couple the ATP hydrolysis to Ca2+ translocation.
Falchetto
R
R
Laboratory of Biochemistry III, Swiss Federal Institute of Technology, ETH, Zürich.
Vorherr
T
T
Carafoli
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary
D002147:Calmodulin; D010446:Peptide Fragments; D010450:Endopeptidases; D002154:Calpain; D000252:Calcium-Transporting ATPases