Membrane Structure of CtrA3, a Copper-transporting P-type-ATPase from Aquifex aeolicus

We have produced and characterized two new copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus, that belong to the family of heavy metal ion-transporting PIB-type ATPases. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH metal-binding motif in TM6 and a histidine-rich N-terminal metal-binding domain. We have cloned both copper pumps, expressed them in Escherichia coli and characterized them functionally. CtrA2 is activated by Ag+ and Cu+ and presumably transports reduced Cu+, while CtrA3 is activated by, and presumably transports, the oxidized copper ion. Both CtrA2 and CtrA3 are thermophilic proteins with an activity maximum at 75 °C. Electron cryomicroscopy of two-dimensional crystals of CtrA3 yielded a projection map at ∼7 Å resolution with density peaks, indicating eight membrane-spanning α-helices per monomer. A fit of the Ca-ATPase structure to the projection map indicates that the arrangement of the six central helices surrounding the ion-binding site in the membrane is conserved, and suggests the position of the two additional N-terminal transmembrane helices that are characteristic of the heavy metal, eight-helix P1B-type ATPases.