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

• Published in: Journal of molecular biology Vol. 378; no. 3; pp. 581 - 595
• Main Authors: Chintalapati, Sivaram, Al Kurdi, Rana, Terwisscha van Scheltinga, Anke C., Kühlbrandt, Werner
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 02.05.2008
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - isolation & purification; Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Aquifex aeolicus; Bacteria - classification; Bacteria - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Cation Transport Proteins - chemistry; Cation Transport Proteins - isolation & purification; Cation Transport Proteins - metabolism; Cell Membrane - chemistry; Cell Membrane - metabolism; Cloning, Molecular; Copper-transporting ATPases; Cryoelectron Microscopy; Crystallography, X-Ray; Cu-ATPase; electron crystallography; Escherichia coli; heavy-metal pumps; Models, Molecular; Molecular Sequence Data; two-dimensional crystallization; TM; Cu-ATPase; SR-CA; Aquifex aeolicus; DDM; heavy-metal pumps; MBD; two-dimensional crystallization; DPPC; electron crystallography
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.01.094

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 P IB-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 P 1B-type ATPases.