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

High-yield heterologous SERCA1 (Ca 2+ 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 His 6 tag at the amino terminus, as compared with a His 6 tag at the carboxyl SERCA terminus, or no tag. The expressed protein was targeted extensively to intracellular membranes. Optimal yield of functional Ca 2+ ATPase corresponded to 10% of total protein, with phosphoenzyme levels, catalytic turnover and Ca 2+ transport identical with those of native SERCA1. This recombinant membrane-bound (detergent-free) enzyme was used for characterization of Ca 2+ binding at the two specific transmembrane sites (ATP-free) by measurements of net charge transfer upon Ca 2+ binding to the protein, yielding cooperative isotherms ( K 1 =5.9±0.5×10 5 M −1 and K 2 =5.7± 0.3×10 6 M −1 ). Non-cooperative binding of only one Ca 2+ , 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 Ca 2+ was increased ( K =4.4±0.2×10 6 M −1 ). This change was due to a p K a shift of site I acidic residues, and to contributions of oxygen functions from empty site II to Ca 2+ binding at site I. No charge movement was observed following E771Q mutation at site I, indicating no Ca 2+ 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 Mg 2+ , the charge movement upon addition of Ca 2+ to WT ATPase was reduced by 50%, while it was reduced by 90% when Ca 2+ was added to the E309Q/A mutants, demonstrating that competitive Mg 2+ binding can occur at site I but not at site II.