Calcium transport by sarcoplasmic reticulum Ca2+-ATPase -: Role of the A domain and its C-terminal link with the transmembrane region

After treatment of sarcoplasmic reticulum Ca2+-ATPase with proteinase K (PK) in the presence of Ca2+ and a protecting non-phosphorylated ligand (e.g. adenosine 5'-(beta,gamma-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE(243) sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca2+ and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADP-sensitive phosphoenzyme, Ca(2)E1P, with occluded Ca2+. The ability of Ca(2)E1P to become converted to the Ca2+-free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca2+. PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage T2' site in the A domain was no longer protected in the absence of Ca2+. It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca2+ ions.