Intramolecular Interactions of the Regulatory Region with the Catalytic Core in the Plasma Membrane Calcium Pump

• Published in: The Journal of biological chemistry Vol. 278; no. 37; pp. 35798 - 35804
• Main Authors: Padányi, Rita, Pászty, Katalin, Penheiter, Alan R., Filoteo, Adelaida G., Penniston, John T., Enyedi, Ágnes
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12-09-2003; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; Calcium-Transporting ATPases - chemistry; Calcium-Transporting ATPases - metabolism; Caspase 3; Caspases - metabolism; Catalysis; Catalytic Domain; Cell Membrane - enzymology; COS Cells; Endoplasmic Reticulum - enzymology; Kinetics; Models, Biological; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sarcoplasmic Reticulum - enzymology; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M305794200

The access of three proteases to their sites of cleavage was used as a measure of regulatory interactions in the plasma membrane Ca2+ pump isoform 4b (PMCA4b). When the proteases could not cut at their sites in the C-terminal regulatory region, the interaction was judged to be tight. This was the case in the absence of Ca2+, when chymotrypsin and caspase cut PMCA only very slowly. Ca2+ accelerated the fragmentation, but the digestion remained incomplete. In the presence of Ca2+ plus calmodulin, the digestion became nearly complete in all cases, indicating a more flexible conformation of the carboxyl terminus in the fully activated state. The acceleration of proteolysis by Ca2+ or Ca2+ plus calmodulin occurred equally at the caspase site upstream of the calmodulin-binding domain and the chymotrypsin and calpain sites downstream of that domain. Replacing Trp1093 (a key residue within the calmodulin-binding domain) with alanine had a much more specific effect, because it exposed only proteolytic sites within the calmodulin-binding domain that had previously been shielded in the native protein. At these sites, both calpain and chymotrypsin cut the Trp1093 → Ala mutant in the absence of calmodulin. These data indicate that, in the auto-inhibited conformation, the calmodulin-binding/auto-inhibitory sequence and the regions both upstream and downstream are in close contact with the catalytic core. Trp1093 plays an essential role not only in stabilizing the Ca2+-calmodulin/calmodulin-binding domain complex but also in the formation or stability of the inhibitory conformation of that domain when it interacts with the catalytic core of PMCA4b.