Structural identification of cation binding pockets in the plasma membrane proton pump

The activity of P-type plasma membrane H+-ATPases is modulated by H+ and cations, with K+ and Ca2+ being of physiological relevance. Using X-ray crystallography, we have located the binding site for Rb+ as a K+ congener, and for Tb3+ and Ho3+ as Ca2+ congeners. Rb+ is found coordinated by a conserved aspartate residue in the phosphorylation domain. A single Tb3+ ion is identified positioned in the nucleotide-binding domain in close vicinity to the bound nucleotide. Ho3+ ions are coordinated at two distinct sites within the H+-ATPase: One site is at the interface of the nucleotidebinding and phosphorylation domains, and the other is in the transmembrane domain toward the extracellular side. The identified binding sites are suggested to represent binding pockets for regulatory cations and a H+ binding site for protons leaving the pump molecule. This implicates Ho3+ as a novel chemical tool for identification of proton binding sites.