Detailed Characterization of the Cooperative Mechanism of Ca²⁺ Binding and Catalytic Activation in the Ca²⁺ Transport (SERCA) ATPase

Abstract

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 Ca(2+) 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 Ca(2+) 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 Ca(2+). 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 Ca(2+) 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 Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) 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 Ca(2+) 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 Ca(2+) binding.

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

“…Similar results were obtained when the ATPase activity of the purified and reconstituted proteins was assayed with strontium (45,46), as displayed in the inset of (Fig. 2B) confirm the limited effect of the ADA mutation on the K m for Ca 2ϩ previously described by Zhang et al (22) for ATPases expressed in COS cells membranes. Conversely, the poor sensitivity to Ca 2ϩ of the hydrolytic activity of the ADA mutant in the presence of detergent, shown in Fig.…”

“…At that time, as we were working with yeast microsomal membranes, we found it necessary to add a solubilizing concentration of C 12 E 8 to the medium during the activity measurements, to suppress the nonspecific hydrolytic activity arising from other proteins present in yeast membranes (this nonspecific activity was making it difficult to unambiguously attribute the small Ca 2ϩ dependent activity, amounting to about 2% of the total activity in the absence of detergent, to expressed SERCA1a). The same ADA mutant enzyme, expressed in COS cells was, however, later reported by Zhang et al (22) to display significant Ca 2ϩ -dependent ATPase activity, up to about 30% of that of the WT enzyme, a level of activity that was detected without any detergent added to the medium (Fig. 4D in Ref.…”

“…Limited Activation of ADA ATPase at Low ATP Concentration-To understand the puzzling discrepancy between the full ATPase activity of the detergent-free ADA mutant and its previously demonstrated (20,22,24) reduced ability for phosphorylation from ATP, we then reinvestigated the ability of the purified and reconstituted ATPases to become phosphorylated from ATP at various calcium concentrations, using an ATP concentration (2 M) low enough to minimize background noise. Panels A and B in Fig.…”

“…The reduction of the cation binding ability of the Ca 2ϩ -ATPase L6 -7 loop after mutation was confirmed by studying a chemically synthesized peptide corresponding to the L6 -7 loop, Gly 808 -Pro 827 , which was found to interact with calcium and to bind lanthanum, in contrast with a related peptide in which an AAA cluster mutation had been introduced (21). In another laboratory both the ADA ATPase mutant and a more conservative related one, D813N,D818N, were found to bind less 45 Ca 2ϩ than WT in the presence of a micromolar Ca 2ϩ concentration (22). The L6 -7 loop was thus suggested to provide an entrance port for calcium, with the negatively charged aspartic residues involved in a pre-binding site (21), and recent computer simulations accepted this view (23).…”

Concerted Conformational Effects of Ca2+and ATP Are Required for Activation of Sequential Reactions in the Ca2+ATPase (SERCA) Catalytic Cycle,

“…Recombinant Ca 2+ ATPase was obtained from COS-1 cells infected with adenovirus vectors carrying chicken WT (33) or mutant cDNA (34). ATP and ADP binding was measured by a filtration method (35)(36)(37) For each experimental sample, 3 ml of ice cold medium were added by fast mixing to 0.3 ml medium containing 0.4 mg SR protein, and filtered after 10 seconds through a 0.65 μm Millipore filter.…”