Intramolecular interactions of the regulatory region with the catalytic core in the plasma membrane calcium pump.
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.
Padányi
Rita
R
National Medical Center, Diószegi utca 64, H-1113 Budapest, Hungary.
Pászty
Katalin
K
Penheiter
Alan R
AR
Filoteo
Adelaida G
AG
Penniston
John T
JT
Enyedi
Agnes
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014162:Transfection
D011994:Recombinant Proteins; D053148:Caspase 3; D020169:Caspases; D000252:Calcium-Transporting ATPases