Biochemical Characterization of CopA, the Escherichia coli Cu(I)-translocating P-type ATPase

Bin Fan,Barry P Rosen

doi:10.1074/jbc.m208490200

Abstract

Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. CopA formed a phosphorylated intermediate with [gamma-(32)P]ATP. Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K(m) for ATP of 0.5 mm. CopA has two N-terminal Cys(X)(2)Cys sequences, Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17), and Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113), and a Cys(479)-Pro-Cys(481) motif in membrane-spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)(2)-Cys motif formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys(479) or Cys(481) resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.

Highlights

The 834-residue CopA copper pump from Escherichia coli [1] is a member of the superfamily of cation-transporting P-type ATPases [2]
We have previously shown that none of the four cysteine residues in the two sequences is required for either copper resistance or transport [18]
Mutations in the two cysteine residues in the Cys-Pro-Cys motif in TM6 resulted in loss of copper resistance, transport, and phosphoenzyme formation, indicating that these cysteines play a more critical role in CopA function than the N-terminal cysteines

Summary

EXPERIMENTAL PROCEDURES

Growth of Cells—Cells of E. coli were grown in Luria-Bertani medium [19] at 37 °C. Ampicillin (100 ␮g/ml), chloramphenicol (30 ␮g/ml), isopropyl-␤-D-thiogalactopyranoside (0.1 mM), 5-bromo-4-chloro-3-indolyl-␤-D-galactosidase (80 ␮g/ml) and L(ϩ)-arabinose (0.0002%) were added as required. To assay inhibition of growth by metal salts, cells were grown overnight in Luria-Bertani medium, diluted 1:100 in the same medium with CuSO4, and incubated for 6 h at 37 °C with shaking. Growth was monitored from the absorbance at 600 nm.

CopA Phosphoenzyme Intermediate

RESULTS

DISCUSSION