Protein Science, Vol 1, Issue 12 1613-1621, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

The calmodulin-binding site of the plasma membrane Ca(2+) pump interacts with the transduction domain of the enzyme

R. FALCHETTO, T. VORHERR and E. CARAFOLI
Laboratory of Biochemistry III, Swiss Federal Institute of Technology (ETH), 8092 Zurich, Switzerland

Calpain proteolysis of the plasma membrane Ca(2+) 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 (3)H-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 Ca(2+) translocation.
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