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J. Biol. Chem., Vol. 279, Issue 52, 54802-54807, December 24, 2004

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Identification of the Transmembrane Metal Binding Site in Cu⁺-transporting P_IB-type ATPases^*

Atin K. Mandal, Ying Yang, Tzipporah M. Kertesz, and José M. Argüello

From the Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609

P_IB-type ATPases have an essential role maintaining copper homeostasis. Metal transport by these membrane proteins requires the presence of a transmembrane metal occlusion/binding site. Previous studies showed that Cys residues in the H6 transmembrane segment are required for metal transport. In this study, the participation in metal binding of conserved residues located in transmembrane segments H7 and H8 was tested using CopA, a model Cu⁺-ATPase from Archaeoglobus fulgidus. Four invariant amino acids in the central portion of H7 (Tyr⁶⁸² and Asn⁶⁸³) and H8 (Met⁷¹¹ and Ser⁷¹⁵) were identified as required for Cu⁺ binding. Replacement of these residues abolished enzyme activity. These proteins did not undergo Cu⁺-dependent phosphorylation by ATP but were phosphorylated by P_i in the absence of Cu⁺. Moreover, the presence of Cu⁺ could not prevent the enzyme phosphorylation by P_i. Other conserved residues in the H7-H8 region were not required for metal binding. Mutation of two invariant Pro residues had little effect on enzyme function. Replacement of residues located close to the cytoplasmic end of H7-H8 led to inactive enzymes. However, these were able to interact with Cu⁺ and undergo phosphorylation. This suggests that the integrity of this region is necessary for conformational transitions but not for ligand binding. These data support the presence of a unique transmembrane Cu⁺ binding/translocation site constituted by Tyr-Asn in H7, Met and Ser in H8, and two Cys in H6 of Cu⁺-ATPases. The likely Cu⁺ coordination during transport appears distinct from that observed in Cu⁺ chaperone proteins or catalytic/redox metal binding sites.

Received for publication, September 21, 2004 , and in revised form, October 18, 2004.

^* This work was supported by National Science Foundation Grant MCM-0235165. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains three additional figures.

To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Rd., Worcester, Massachusetts 01609. Tel.: 508-831-5326; Fax: 508-831-5933; E-mail: arguello{at}wpi.edu.

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