Copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B are evolutionarily conserved polytopic membrane proteins with essential roles in human physiology. The Cu-ATPases are expressed in most tissues, and their transport activity is crucial for central nervous system development, liver function, connective tissue formation, and many other physiological processes. The loss of ATP7A or ATP7B function is associated with severe metabolic disorders, Menkes disease, and Wilson disease. In cells, the Cu-ATPases maintain intracellular copper concentration by transporting copper from the cytosol across cellular membranes. They also contribute to protein biosynthesis by delivering copper into the lumen of the secretory pathway where metal ion is incorporated into copper-dependent enzymes. The biosynthetic and homeostatic functions of Cu-ATPases are performed in different cell compartments; targeting to these compartments and the functional activity of Cu-ATPase are both regulated by copper. In recent years, significant progress has been made in understanding the structure, function, and regulation of these essential transporters. These studies raised many new questions related to specific physiological roles of Cu-ATPases in various tissues and complex mechanisms that control the Cu-ATPase function. This review summarizes current data on the structural organization and functional properties of ATP7A and ATP7B as well as their localization and functions in various tissues, and discusses the current models of regulated trafficking of human Cu-ATPases.

Function and Regulation of Human Copper-Transporting ATPases

The transition or d-block metal ions manganese, iron, cobalt, nickel, copper, zinc, and to a more specialized degree, molybdenum, tungsten and vanadium, have been shown to be important for biological systems. These metal ions are ubiquitously found in nature, nearly exclusively as constituents of proteins.1 The unique properties of metal ions have been exploited by nature to perform a wide range of tasks. These include roles as structural components of biomolecules, as signaling molecules, as catalytic cofactors in reversible oxidation-reduction and hydrolytic reactions, and in structural rearrangements of organic molecules and electron transfer chemistry.1 Indeed, metal ions play critical roles in the cell that cannot be performed by any other entity, and are therefore essential for all of life. However, an individual metal ion is capable of performing only one or a few of these functions, but certainly not all; as a result, nature has evolved mechanisms to effectively distinguish one metal from another. The coordination chemistry of metal ion-protein complexes is fundamental to this biological discrimination, and is largely the focus of this review.

1.1. Metal Ion Homeostasis
Extensive regulatory and protein-coding machinery is devoted to maintaining the “homeostasis” of biologically required metal ions and underscores the essentiality of this process for cell viability. Homeostasis is defined as the maintenance of an optimal bioavailable concentration, mediated by the balancing of metal uptake and intracellular trafficking with efflux/storage processes so that the needs of the cell for that metal ion is met, i.e., the “right” metal is inserted into the “right” macromolecule at the appropriate time.2,3 Just as a scarcity of a particular metal ion induces a stress response that can lead to reprogramming of cellular metabolism to minimize the consequences of depletion of a particular metal ion, e.g., zinc in ribosome biogenesis4 or Cu vs. Fe in photosynthesis by Synechocystis,5 too much of the same metal ion can also be toxic to a cell or organism.

Metal homeostasis is governed by the formation of specific protein-metal coordination complexes used to effect uptake, efflux, intracellular trafficking within compartments, and storage (Figure 1). How metal ions move to and from their target destinations in the active site of a metalloenzyme or as a structural component of biomolecules also contributes to intracellular metal homeostasis (Figure 1). Metal transporters move metal ions or small molecule-metal chelates across otherwise impermeable barriers in a directional fashion, and most of these are integral membrane proteins embedded in the inner or plasma membrane (Figure 1). Specialized protein chelators designated metallochaperones traffic metals within a particular cellular compartment, e.g., the periplasm or the cytosol, and function to “hold” the metal in such a way that it can be readily transferred to an appropriate acceptor protein. This intermolecular transfer is known or is projected to occur through transiently formed, specific protein-protein complexes that mediate coordinated intermolecular metal ligand exchange. Metallochaperones have been described for copper,6-9 nickel10 and iron-sulfur protein biogenesis,11 and recent work suggests that the periplasmic Zn(II) binding protein, YodA, has characteristics consistent with a role as a zinc chaperone in E. coli (Figure 1).12 Salient features of these chaperones are discussed in more detail in the context of acquisition and efflux of individual metal ions (Section 2). Finally, specialized transcriptional regulatory proteins, termed metalloregulatory or metal sensor proteins, control the expression of genes encoding these proteins that establish metal homeostasis in response to either metal deprivation or overload (Section 3).



Figure 1

Schematic metal homeostasis models for iron, zinc and manganese, copper, nickel and cobalt, shown specifically in gram-negative bacteria. Homeostasis of molybdate and tungstate oxyanions are not shown, due primarily to a lack of knowledge of these systems, ...



A hypothesis that emerges is that in order to effect the cellular homeostasis of a particular metal ion, each component of the homeostasis machinery (Figure 1) must be selective for that metal ion under the prevailing conditions, to the exclusion of all others.13 Furthermore, individual systems must be “tuned” such that the affinity or sensitivity of each component is well-matched, either to coordinate gene expression by pairs of metal sensor proteins that coordinately shut off uptake and up-regulate efflux or detoxification systems, or to facilitate vectorial transport from metal donor to metal acceptor target protein in a metal trafficking pathway in the cell (Figure 1).14-16

Coordination chemistry of bacterial metal transport and sensing.

Trafficking of the copper-ATPases, ATP7A and ATP7B: Role in copper homeostasis

1.1. Background
The use of copper in biological systems coincides with the advent of an oxygen atmosphere about 1.7 billion years ago. The presence of O2 both allowed the oxidation of insoluble Cu(I) to the more soluble and bioavailable Cu(II) and led to the requirement for a redox active metal with potentials in the 0-800 mV range. Not only did copper meet this need, but the oxidation of Fe(II) to the insoluble Fe(III) form rendered the use of iron more energetically expensive.1-5 As a result, copper plays a key role in many proteins that react with O2. Generally, O2-reactive centers are mononuclear (type 2), dinuclear (type 3), or trinuclear (type 2 and type 3). Well studied mononuclear copper enzymes include the monooxygenases dopamine-β-hydroxylase and peptidylglycine α-hydroxylating monooxygenase as well as oxidases that also contain organic cofactors, such as amine, galactose, and lysyl oxidases.6 Dinuclear copper proteins include the O2 carrier hemocyanin and enzymes like tyrosinase and catechol oxidase.7 Copper also plays a key role in numerous electron transfer proteins. Mononuclear type 1 (blue copper) centers are found in proteins such as plastocyanin and azurin.8 The multicopper oxidases like laccase, ascorbate oxidase, and ceruloplasmin contain both a catalytic trinuclear type 2/type 3 site and an electron transfer type 1 site.9,10 The classification of copper centers into types is derived from optical and electron paramagnetic resonance (EPR) spectroscopic properties, and there are some notable exceptions, including the cysteine-bridged dinuclear CuA electron transfer site in cytochrome c oxidase11 and nitrous oxide reductase, the tetranuclear catalytic CuZ center in nitrous oxide reductase,12 and the proposed catalytic copper center in particulate methane monooxygenase.13-15

The same redox properties that render copper useful in all these metalloproteins can lead to oxidative damage in cells. Reaction of Cu(I) with hydrogen peroxide and re-reduction of Cu(II) by superoxide via Fenton and Haber-Weiss chemistry yields hydroxyl radicals that can damage proteins, lipids, and nucleic acids.16 Thus, intracellular copper concentrations must be controlled such that copper ions are provided to essential enzymes, but do not accumulate to deleterious levels. In humans, deficiencies in copper metabolism are linked to diseases such as Menkes syndrome, Wilson disease, prion diseases, and Alzheimer’s disease.17 Several classes of proteins, including membrane transporters,18-20 metallochaperones,21,22 and metalloregulatory proteins,23,24 are implicated in copper homeostasis. These proteins have two functions. First, they ensure that copper is provided to the correct proteins and cellular compartments for necessary activities. Second, these proteins detoxify excess copper. Just as copper-containing proteins and enzymes are found in all kingdoms of life, members of these groups of homeostatic proteins are also widespread,5 and have been structurally and biochemically characterized from eukaryotes and prokaryotes.

Structural biology of copper trafficking.

Platinum-based antitumor agents have been effective in the treatments of many human malignancies but the ultimate success of these agents is often compromised by development of drug resistance. One mechanism associated with resistance to platinum drugs is reduced intracellular accumulation owing to impaired drug intake, enhanced outward transport, or both. Mechanisms for transporting platinum drugs were not known until recent demonstrations that import and export transporters involved in maintenance copper homeostasis are also involved in the transport of these drugs. Ctr1, the major copper influx transporter, has been convincingly demonstrated to transport cisplatin and its analogues, carboplatin, and oxaliplatin. Evidence also suggests that the two copper efflux transporters ATP7A and ATP7B regulate the efflux of cisplatin. These observations are intriguing, because conventional thinking of the inorganic physiologic chemistry of cisplatin and copper is quite different. Hence, understanding the underlying mechanistic aspects of these transporters is critically important. While the mechanisms by which hCtr1, ATP7A and ATP7B transport copper ions have been studied extensively, very little is known about the mechanisms by which these transporters shuffle platinum-based antitumor agents. This review discusses the identification of copper transporters as platinum drug transporters, the structural-functional and mechanistic aspects of these transporters, the mechanisms that regulate their expression, and future research directions that may eventually lead to improved efficacy of platinum-based-based drugs in cancer chemotherapy through modulation of their transporters’ activities.

The roles of copper transporters in cisplatin resistance.

Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein.

Human Wilson protein is a copper-transporting ATPase located in the secretory pathway possessing six N-terminal metal-binding domains. Here we focus on the function of the metal-binding domains closest to the vesicular portion of the copper pump, i.e., domain 4 (WLN4), and a construct of domains 5 and 6 (WLN5–6). For comparison purposes, some experiments were also performed with domain 2 (WLN2). The solution structure of apoWLN5–6 consists of two ferredoxin folds connected by a short linker, and 15N relaxation rate measurements show that it behaves as a unit in solution. An NMR titration of apoWLN5–6 with the metallochaperone Cu(I)HAH1 reveals no complex formation and no copper exchange between the two proteins, whereas titration of Cu(I)HAH1 with WLN4 shows the formation of an adduct that is in fast exchange on the NMR time scale with the isolated protein species as confirmed by 15N relaxation data. A similar interaction is also observed between Cu(I)HAH1 and WLN2; however, the relative amount of the adduct in the protein mixture is lower. An NMR titration of apoWLN5–6 with Cu(I)WLN4 shows copper transfer, first to WLN6 then to WLN5, without the formation of an adduct. Therefore, we suggest that WLN4 and WLN2 are two acceptors of Cu(I) from HAH1, which then somehow route copper to WLN5–6, before the ATP-driven transport of copper across the vesicular membrane.

https://www.pnas.org/content/pnas/103/15/5729.full.pdf

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake

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Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein.

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake