Characterization of a Thermophilic P-type Ag+/Cu+-ATPase from the ExtremophileArchaeoglobus fulgidus

Abstract

The thermophilic, sulfur metabolizing Archaeoglobus fulgidus contains two genes, AF0473 and AF0152, encoding for PIB-type heavy metal transport ATPases. In this study, we describe the cloning, heterologous expression, purification, and functional characterization of one of these ATPases, CopA (NCB accession number AAB90763), encoded by AF0473. 50 ‫؍‬ 24 M). This is the first Ag ؉ /Cu ؉ -ATPase expressed and purified in a functional form. Thus, it provides a model for structurefunctional studies of these transporters. Moreover, its characterization will also contribute to an understanding of thermophilic ion transporters.

Mechanism of Cu+-transporting ATPases: Soluble Cu+chaperones directly transfer Cu+to transmembrane transport sites

“…5. In all experiments, these proteins were in soluble form stabilized in mixed 0.01% dodecyl-␤-D-maltoside (DDM), 0.01% asolectin micelles.…”

“…A. fulgidus CopA, CopZ, and C 27,30,751,754 A CopA cDNAs were obtained as described (5,16,32). A construct encoding for truncated CopA extending from Gly 80 to Gly 736 and therefore lacking the N-MBD and C-MBD (⌬N,C-CopA) was obtained by PCR by using CopA as template.…”

“…The subsequent conformational change allows metal deocclusion and release to the extracellular (vesicular/luminal) compartment followed by enzyme dephosphorylation and return to the E1 form (10). Functional studies of various Cu ϩ -ATPases have characterized the Cu ϩ transport, Cu ϩ -dependent ATPase activity, phosphorylation, and dephosphorylation partial reactions (5,(13)(14)(15)(16)(17)(18)(19).Cu ϩ -ATPases consist of eight transmembrane segments, two large cytosolic loops comprising the A-domain and the ATP-BD, and regulatory metal-binding domains (MBDs) in their N terminus (6, 8-10, 20, 21) (Fig. 1).…”

The Mechanism of Cu+ Transport ATPases

“…AfCopZ wild type and mutant protein expression was induced for 3 h by the addition of 1 mM isopropyl-␤-D-thiogalactopyranoside. Purification of membrane and soluble proteins were carried out as described (10,18,34,38). Solubilized lipid/detergent micellar forms of ⌬N,C-CopA proteins were stored in buffer containing 25 mM Hepes (pH 8.0), 500 mM NaCl, 10 mM ascorbic acid, 0.01% n-dodecyl-␤-D-maltopyranoside (DDM), and 0.01% asolectin until use.…”

“…The Archaeoglobus fulgidus CopA-CopZ provides a framework to test these ideas, because this Cu ϩ -ATPase (AfCopA) and its corresponding Cu ϩ chaperone (AfCopZ) have served as models for previous mechanistic studies (11,15,16,18,34). The description of their interaction provided a basis for our current understanding of Cu ϩ access to transmembrane transporters (10,24).…”