Expression, isolation, and crystallization of the catalytic domain of CopB, a putative copper transporting ATPase from the thermoacidophilic archaeon Sulfolobus solfataricus.
The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.
Deigweiher
Katrin
K
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum.
Drell
Theodore L
TL
Prutsch
Alexander
A
Scheidig
Axel J
AJ
Lübben
Mathias
M
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology / Q000235:genetics; D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D003460:Crystallization / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019848:Gene Expression Regulation, Archaeal / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D019843:Archaeal Proteins; D027682:Cation Transport Proteins; C489503:CopB protein, Archaea; D000079922:Copper Transport Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases