High-yield Heterologous Expression of Wild Type and Mutant Ca2+ ATPase: Characterization of Ca2+ Binding Sites by Charge Transfer

High-yield heterologous SERCA1 (Ca2+ ATPase) expression was obtained in COS-1 cells infected with recombinant adenovirus vector (rAdSERCA). Higher transcription and expression were obtained in the presence of a His6 tag at the amino terminus, as compared with a His6 tag at the carboxyl SERCA terminus, or no tag. The expressed protein was targeted extensively to intracellular membranes. Optimal yield of functional Ca2+ ATPase corresponded to 10% of total protein, with phosphoenzyme levels, catalytic turnover and Ca2+ transport identical with those of native SERCA1. This recombinant membrane-bound (detergent-free) enzyme was used for characterization of Ca2+ binding at the two specific transmembrane sites (ATP-free) by measurements of net charge transfer upon Ca2+ binding to the protein, yielding cooperative isotherms (K1 = 5.9 ± 0.5 × 105 M−1 and K2 = 5.7 ± 0.3 × 106 M−1). Non-cooperative binding of only one Ca2+, and loss of ATPase activation, were observed following E309 mutation at site II. On the other hand, as a consequence of the site II mutation, the affinity of site I for Ca2+ was increased (K = 4.4 ± 0.2 × 106 M−1). This change was due to a pKa shift of site I acidic residues, and to contributions of oxygen functions from empty site II to Ca2+ binding at site I. No charge movement was observed following E771Q mutation at site I, indicating no Ca2+ binding to either site. Therefore, calcium occupancy of site I is required to trigger cooperative binding to site II and catalytic activation. In the presence of millimolar Mg2+, the charge movement upon addition of Ca2+ to WT ATPase was reduced by 50%, while it was reduced by 90% when Ca2+ was added to the E309Q/A mutants, demonstrating that competitive Mg2+ binding can occur at site I but not at site II.