José M. Argüello

The structure and function of heavy metal transport P1B-ATPases

P1B-type ATPases transport heavy metals (Cu+, Cu2+, Zn2+, Co2+, Cd2+, Pb2+) across membranes. Present in most organisms, they are key elements for metal homeostasis. P1B-type ATPases contain 6-8 transmembrane fragments carrying signature sequences in segments flanking the large ATP binding cytoplasmic loop. These sequences made possible the differentiation of at least four P1B-ATPase subgroups with distinct metal selectivity: P1B-1: Cu+, P1B-2: Zn2+, P1B-3: Cu2+, P1B-4: Co2+. Mutagenesis of the invariant transmembrane Cys in H6, Asn and Tyr in H7 and Met and Ser in H8 of the Archaeoglobus fulgidus Cu+-ATPase has revealed that their side chains likely coordinate the metals during transport and constitute a central unique component of these enzymes. The structure of various cytoplasmic domains has been solved. The overall structure of those involved in enzyme phosphorylation (P-domain), nucleotide binding (N-domain) and energy transduction (A-domain), appears similar to those described for the SERCA Ca2+-ATPase. However, they show different features likely associated with singular functions of these proteins. Many P1B-type ATPases, but not all of them, also contain a diverse arrangement of cytoplasmic metal binding domains (MBDs). In spite of their structural differences, all N- and C-terminal MBDs appear to control the enzyme turnover rate without affecting metal binding to transmembrane transport sites. In addition, eukaryotic Cu+-ATPases have multiple N-MBD regions that participate in the metal dependent targeting and localization of these proteins. The current knowledge of structure-function relationships among the different P1B-ATPases allows for a description of selectivity, regulation and transport mechanisms. Moreover, it provides a framework to understand mutations in human Cu+-ATPases (ATP7A and ATP7B) that lead to Menkes and Wilson diseases.

Identification of ion-selectivity determinants in heavy-metal transport P1B-type ATPases.

P1B-type ATPases transport a variety of metals (Cd2+, Zn2+, Pb2+, Co2+, Cu2+, Ag+, Cu+) across biomembranes. Characteristic sequences CP[C/H/S] in transmembrane fragment H6 were observed in the putative transporting metal site of the founding members of this subfamily (initially named CPx-ATPases). In spite of their importance for metal homeostasis and biotolerance, their mechanisms of ion selectivity are not understood. Studies of better-characterized PII-type ATPases (Ca-ATPase and Na,K-ATPase) have identified three transmembrane segments that participate in ion binding and transport. Testing the hypothesis that metal specificity is determined by conserved amino acids located in the equivalent transmembrane segments of P1B-type ATPases (H6, H7, and H8), 234 P1B-ATPase protein sequences were analyzed. This showed that although H6 contains characteristic CPX or XPC sequences, conserved amino acids in H7 and H8 provide signature sequences that predict the metal selectivity in each of five P1B-ATPase subgroups identified. These invariant amino acids contain diverse side chains (thiol, hydroxyl, carbonyl, amide, imidazolium) that can participate in transient metal coordination during transport and consequently determine the particular metal selectivity of each enzyme. Each subgroup shares additional structural characteristics such as the presence (or absence) of particular amino-terminal metal-binding domains and the number of putative transmembrane segments. These differences suggest unique functional characteristics for each subgroup in addition to their particular metal specificity.

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

As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in Cu+-ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu+ does not access Cu+-ATPases in a free (hydrated) form but is bound to a chaperone protein. Cu+ transfer from Cu+ chaperones to regulatory cytoplasmic metal-binding domains (MBDs) present in these ATPases has been described, but there is no evidence of a proposed subsequent Cu+ movement from the MBDs to the TM-MBS. Alternatively, we postulate the parsimonious Cu+ transfer by the chaperone directly to TM-MBS. Testing both models, the delivery of Cu+ by Archaeoglobus fulgidus Cu+ chaperone CopZ to the corresponding Cu+-ATPase, CopA, was studied. As expected, CopZ interacted with and delivered the metal to CopA MBDs. Cu+-loaded MBDs, acting as metal donors, were unable to activate CopA or a truncated CopA lacking MBDs. Conversely, Cu+-loaded CopZ activated the CopA ATPase and CopA constructs in which MBDs were rendered unable to bind Cu+. Furthermore, under nonturnover conditions, CopZ transferred Cu+ to the TM-MBS of a CopA lacking MBDs. These data are consistent with a model where MBDs serve a regulatory function without participating in metal transport and the chaperone delivers Cu+ directly to transmembrane transport sites of Cu+-ATPases.

Arabidopsis HMA2, a divalent heavy metal-transporting PIB-type ATPase, is involved in cytoplasmic Zn2+ homeostasis

PIB-type ATPases transport heavy metal ions (Cu+, Cu2+, Zn2+, Cd2+, Co2+, etc.) across biological membranes. Several members of this subfamily are present in plants. Higher plants are the only eukaryotes where putative Zn2+-ATPases have been identified. We have cloned HMA2, a PIB-ATPase present in Arabidopsis (Arabidopsis thaliana), and functionally characterized this enzyme after heterologous expression in yeast (Saccharomyces cerevisiae). HMA2 is a Zn2+-dependent ATPase that is also activated by Cd2+ and, to a lesser extent, by other divalent heavy metals (Pb2+, Ni2+, Cu2+, and Co2+). The enzyme forms an acid-stable phosphorylated intermediate and is inhibited by vanadate. HMA2 interacts with Zn2+ and Cd2+ with high affinity (Zn2+ K1/2 = 0.11 ± 0.03 μm and Cd2+ K1/2 = 0.031 ± 0.007 μm). However, its activity is dependent on millimolar concentrations of Cys in the assay media. Zn2+ transport determinations indicate that the enzyme drives the outward transport of metals from the cell cytoplasm. Analysis of HMA2 mRNA suggests that the enzyme is present in all plant organs and transcript levels do not change in plants exposed to various metals. Removal of HMA2 full-length transcript results in Zn2+ accumulation in plant tissues. hma2 mutant plants also accumulate Cd2+ when exposed to this metal. These results suggest that HMA2 is responsible for Zn2+ efflux from the cells and therefore is required for maintaining low cytoplasmic Zn2+ levels and normal Zn2+ homeostasis.

Distinct functional roles of homologous Cu+ efflux ATPases in Pseudomonas aeruginosa

In bacteria, most Cu+‐ATPases confer tolerance to Cu by driving cytoplasmic metal efflux. However, many bacterial genomes contain several genes coding for these enzymes suggesting alternative roles. Pseudomonas aeruginosa has two structurally similar Cu+‐ATPases, CopA1 and CopA2. Both proteins are essential for virulence. Expressed in response to high Cu, CopA1 maintains the cellular Cu quota and provides tolerance to this metal. CopA2 belongs to a subgroup of ATPases that are expressed in association with cytochrome oxidase subunits. Mutation of copA2 has no effect on Cu toxicity nor intracellular Cu levels; but it leads to higher H2O2 sensitivity and reduced cytochrome oxidase activity. Mutation of both genes does not exacerbate the phenotypes produced by single‐gene mutations. CopA1 does not complement the copA2 mutant strain and vice versa, even when promoter regions are exchanged. CopA1 but not CopA2 complements an Escherichia coli strain lacking the endogenous CopA. Nevertheless, transport assays show that both enzymes catalyse cytoplasmic Cu+ efflux into the periplasm, albeit CopA2 at a significantly lower rate. We hypothesize that their distinct cellular functions could be based on the intrinsic differences in transport kinetic or the likely requirement of periplasmic partner Cu‐chaperone proteins specific for each Cu+‐ATPase.

Structure of the ATP Binding Domain from the Archaeoglobus fulgidus Cu+-ATPase

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 Å resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.

Asp804 and Asp808 in the transmembrane domain of the Na,K-ATPase alpha subunit are cation coordinating residues.

The functional roles of Asp804 and Asp808, located in the sixth transmembrane segment of the Na,K-ATPase α subunit, were examined. Nonconservative replacement of these residues yielded enzymes unable to support cell viability. Only the conservative substitution, Ala808 → Glu, was able to maintain the essential cation gradients (Van Huysse, J. W., Kuntzweiler, T. A., and Lingrel, J. B (1996) FEBS Lett. 389, 179-185). Asp804 and Asp808 were replaced by Ala, Asn, and Glu in the sheep α1 subunit and expressed in a mouse cell line where [3H]ouabain binding was utilized to probe the exogenous proteins. All of the heterologous proteins were targeted into the plasma membrane, bound ouabain and nucleotides, and adopted E1Na, E1ATP, and E2P conformations. K+ competition of ouabain binding to sheep α1 and Asp808 → Glu enzymes displayed IC50 values of 4.11 mM (nHill = 1.4) and 23.8 mM (nHill = 1.6), respectively. All other substituted proteins lacked this K+-ouabain antagonism, e.g. 150 mM KCl did not inhibit ouabain binding. Na+ antagonized ouabain binding to all the expressed isoforms, however, the proteins carrying nonconservative substitutions displayed reduced Hill coefficients (nHill ≤ 2.0) compared to the control (nHill ≤ 2.8). Therefore, Asp804 and Asp808 of the Na,K-ATPase are required for normal Na+ and K+ transport, possibly coordinating these cations during transport.

Structure of the Two Transmembrane Cu+ Transport Sites of the Cu+-ATPases

Cu+-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu+ within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu+ transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu+. However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu+-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu+ (Site I Ka = 1.3 fm–1, Site II Ka = 1.1 fm–1), in conjunction with reversible direct Cu+ transfer from chaperones, points to a transport mechanism where backward release of free Cu+ to the cytoplasm is largely prevented.

The transport mechanism of bacterial Cu+-ATPases: distinct efflux rates adapted to different function

Cu+-ATPases play a key role in bacterial Cu+ homeostasis by participating in Cu+ detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P1B-1 type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu+ with high affinity in a trigonal planar geometry. The cytoplasmic Cu+ chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu+ is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent transport studies have shown that all Cu+-ATPases drive cytoplasmic Cu+ efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu+-efflux pumps responsible for Cu+ tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments.

Archaeoglobus fulgidus CopB Is a Thermophilic Cu2+-ATPase FUNCTIONAL ROLE OF ITS HISTIDINE-RICH N-TERMINAL METAL BINDING DOMAIN

P1B-type ATPases transport heavy metal ions across cellular membranes. Archaeoglobus fulgidus CopB is a member of this subfamily. We have cloned, expressed in Escherichia coli, and functionally characterized this enzyme. CopB and its homologs are distinguished by a metal binding sequence Cys-Pro-His in their sixth transmembrane segment (H6) and a His-rich N-terminal metal binding domain (His-N-MBD). CopB is a thermophilic protein active at 75 °C and high ionic strength. It is activated by Cu2+ with high apparent affinity (K1/2 = 0.28 μm) and partially by Cu+ and Ag+ (22 and 55%, respectively). The higher turnover was associated with a faster phosphorylation rate in the presence of Cu2+. A truncated CopB lacking the first 54 amino acids was constructed to characterize the His-N-MBD. This enzyme showed reduced ATPase activity (50% of wild type) but no changes in metal selectivity, ATP dependence, or phosphorylation levels. However, a slower rate of dephosphorylation of the E2P(Cu2+) form was observed for truncated CopB. The data suggest that the presence of the His residue in the putative transmembrane metal binding site of CopB determines a selectivity for this enzyme that is different for that observed in Cu+/Ag+-ATPases carrying a Cys-Pro-Cys sequence. The His-NMBD appears to have a regulatory role affecting the metal transport rate by controlling the metal release/dephosphorylation rates.