Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10−15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca²⁺ binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P_i. Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca²⁺ required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca²⁺. The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca²⁺ binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca²⁺ occupancy of site 1, followed by transition to a conformation (E‘) sensitive to Ca²⁺ inhibition of enzyme phosphorylation by P_i, but still unable to utilize ATP. The E‘ conformation accepts the second Ca²⁺ on site 2, producing then a conformation (E‘ ‘) which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca²⁺ affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain) produces total inhibition of ATP utilization and enzyme phosphorylation by P_i, without a significant effect on Ca²⁺ binding.

Publications with shared reference:

0 publications found