Identification of Calcium‐Binding Sites in the Nucleotide Binding Domain of the Plasma Membrane H ⁺ ‐ATPase From Saccharomyces cerevisiae

In Saccharomyces cerevisiae , calcium signaling mediates the activation of the plasma membrane H ⁺ ‐ATPase by glucose. Proton pumping activity and calcium influx appear to be closely linked, since the H ⁺ ‐ATPase interacts physically with Mid1, which is a subunit of the voltage‐gated Ca ²⁺ channel (VGCC). Thus, calcium immediately finds the entire H ⁺ ‐ATPase cytoplasmic structure during influx, where potential interactions may occur. In this work, several calcium‐binding sites were suggested in the nucleotide‐binding domain (N‐domain) through three‐dimensional (3D) structural analysis and molecular dynamics simulation (MDS). The calcium‐binding sites consisted mainly of Asp and Glu residues that displayed bidentate coordination geometry. Calcium binding was confirmed in vitro through intrinsic fluorescence quenching of a recombinant N‐domain protein and energy‐transfer‐sensitized Tb ³⁺ luminescence. Tb ³⁺ binding to the N‐domain was tested under different experimental conditions, which confirmed the interaction. The calcium ion binds to the H ⁺ ‐ATPase N‐domain and is likely directly involved in modulating enzyme activity.