Expression, isolation, and crystallization of the catalytic domain of CopB, a putative copper transporting ATPase from the thermoacidophilic archaeon Sulfolobus solfataricus

J Bioenerg Biomembr. 2004 Feb;36(1):151-9. doi: 10.1023/b:jobb.0000019607.05233.4c.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases
  • Amino Acid Sequence
  • Archaea / enzymology
  • Archaea / genetics
  • Archaeal Proteins / biosynthesis*
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / isolation & purification
  • Catalysis
  • Cation Transport Proteins
  • Copper Transport Proteins
  • Crystallization / methods
  • Enzyme Activation
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Gene Expression Regulation, Archaeal / physiology
  • Gene Expression Regulation, Enzymologic / physiology
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Structure, Tertiary
  • Proton-Translocating ATPases / biosynthesis*
  • Proton-Translocating ATPases / chemistry*
  • Proton-Translocating ATPases / genetics
  • Proton-Translocating ATPases / isolation & purification
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Sequence Homology, Amino Acid
  • Sulfolobus / enzymology*
  • Sulfolobus / genetics
  • Temperature

Substances

  • Archaeal Proteins
  • Cation Transport Proteins
  • CopB protein, Archaea
  • Copper Transport Proteins
  • Recombinant Proteins
  • Adenosine Triphosphatases
  • Proton-Translocating ATPases