Functional roles of metal binding domains of the Archaeoglobus fulgidus Cu(+)-ATPase CopA.
CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) or Ag(+) [Mandal et al. (2002) J. Biol. Chem. 277, 7201-7208]. This, as other P(IB)-ATPases, is characterized by a putative metal binding sequence (C(380)PC(382)) in its sixth transmembrane fragment and cytoplasmic metal binding sequences in its NH(2)- and COOH-terminal ends (C(27)AMC(30) and C(751)HHC(754)). Using isolated CopA, we have studied the functional role of these three putative metal binding domains. Replacement of transmembrane Cys residues by Ala results in nonfunctional enzymes that are unable to hydrolyze ATP. However, the CPC --> APA substituted enzyme binds ATP, indicating its correct folding and suggesting that enzyme turnover is prevented by the lack of metal binding to the transmembrane site. Replacement of C-terminal Cys by Ala (C(751,754)A) has no significant effect on ATPase activity, enzyme phosphorylation, apparent binding affinities of ligands, or E1-E2 equilibrium. In contrast, replacement of Cys in the N-terminal metal binding domain (N-MBD) (C(27,30)A) leads to 40% reduction in enzyme turnover. The C(27,30)A enzyme binds Cu(+), Ag(+), and ATP with the same high apparent affinities as the wild-type CopA. Evidence that N-MBD disruption has no effect on the E1-E2 equilibrium is provided by the normal interaction of ATP acting with low affinity and the unaffected IC(50) for vanadate inhibition observed in the C(27,30)A-substituted enzyme. However, replacement C(27,30)A slowed the dephosphorylation of the E2P(metal) form of the enzyme, suggesting a reduction in the rate of metal release. Other investigators have shown the Cu-dependent interaction of isolated N-MBDs from the Wilson disease Cu-ATPase with the ATP binding cytoplasmic domain [Tsivkovskii et al. (2001) J. Biol. Chem. 276, 2234-2242]. Therefore, the data suggest a regulatory mechanism in which the Cu-dependent N-MBD/ATP binding domain interaction would accelerate cation release, the enzyme rate-limiting step, and consequently Cu(+) transport.
Mandal
Atin K
AK
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Road, Worcester, Massachusetts 01609, USA.
Argüello
José M
JM
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019611:Archaeoglobus fulgidus / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D000073840:Copper-Transporting ATPases; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008024:Ligands; D008670:Metals / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; C071263:CopA protein, Bacteria; D008024:Ligands; D008670:Metals; D014638:Vanadates; D003300:Copper; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases; D003545:Cysteine