Fifty years ago, the neurologist John Menkes described an inherited brain disease in children that was associated with brittle, white, kinky hair. A clue to the origin of the fatal condition came from sheep grazing on copper-deficient pasture in Australia: the animals' steely wool looked like the hair of children with Menkes disease. In both cases, the explanation was found to be inactive copper-dependent enzymes (cuproenzymes). More precisely, affected children have a faulty protein1 known as the P1B-type ATPase ATP7A (often called the Menkes ATPase), an enzyme that pumps copper ions through membranes and has a vital role in distributing copper around the body.

On page 59 of this issue, Gourdon et al.2 present the much-anticipated crystal structure of a P1B-type ATPase, and reveal a platform in the protein that creates a loading site for copper. A helix peculiar to P1B-type ATPases bends to form the platform, and the authors find that amino-acid residues at the bend of this helix are often mutated in the ATP7A protein of patients with Menkes disease (Fig. 1). More broadly, the structure provides information about the architecture of the P1B-type ATPase family of metal-transporter proteins, which are ubiquitous in bacteria, plants and animals.

Figure 1: A copper-exporting enzyme.
figure 1

a, Gram-negative bacteria use P1B-type ATPase enzymes (such as LpCopA in L. pneumophila, shown) to pump copper ions from the cytosol across their inner membrane and into the periplasm. In many organisms, metallochaperone molecules deliver the copper to the enzyme. b, Gourdon and colleagues' crystal structure2 of LpCopA reveals a platform formed by a kink in an α-helix of the protein. The structure suggests that copper's pathway through the protein (arrows) involves three sets of binding sites (numbered 1 to 3), the first of which is on the platform. Copper is probably delivered to the platform site by a metallochaperone (as shown), or by a mobile heavy-metal-binding domain (HMBD) of LpCopA. The HMBD might regulate the activity of LpCopA by interacting with the protein's actuator domain (A) or nucleotide-binding domain (N), or, indirectly, with the phosphorylation domain (P). By analogy with the LpCopA structure, the authors deduce that certain mutations of the ATPase ATP7A that are associated with Menkes disease cluster around the kink of the platform-forming helix (red star).

Cells handle copper with care. Most cuproenzymes are confined to intracellular compartments or are expelled from cells to perform roles in extracellular fluids and matrices. Other proteins known as metallochaperones 'hand-deliver' copper to where it is needed, relinquishing their load only to those proteins with which they specifically interact and which bind copper most tightly3. Proteins such as the Menkes ATPase pump copper from cells to prevent an excess of the metal from accumulating inside. They also pump copper from the cytosol into intracellular compartments where the metal is needed as a cofactor for cuproenzymes4, as well as into compartments that carry copper and cuproenzymes out of cells. When the Menkes ATPase fails, rather than being loaded into the bloodstream, copper becomes lodged inside cells lining the intestine. This, in turn, leads to a defective supply of copper cofactor for cuproenzymes5.

Gourdon and colleagues' structure2 of LpCopA — a P1B-type ATPase from the bacterium Legionella pneumophila — is relevant to an eclectic range of sub-disciplines. For example, plant P1B-type ATPases deliver copper into the compartments of chloroplasts, where the metal ions conduct electrons in the process that converts light into chemical energy (the first stage of photosynthesis)6. Elevated levels of the Menkes ATPase have been linked to the progression of Alzheimer's disease, and a related protein, ATP7B, limits the efficacy of the anticancer drug cisplatin. P1B-type ATPases are also vital weapons in the immune system's battle against pathogenic bacteria7: when immune cells engulf invading microbes, they attack them with bactericidal copper pumped by Menkes ATPases; the bacteria use their own P1B-type ATPases in defence. Microbial P1B-type ATPases also counter the copper used as a fungicide in vineyards, and as a disinfectant in public water systems that can harbour Legionnaires' disease.

Because copper is bound and/or chaperoned in cells, there has been interest in understanding how P1B-type ATPases acquire copper from the cytosol. These proteins have soluble heavy-metal-binding domains (HMBDs, Fig. 1) that protrude into the cytosol and interact with copper chaperones electrostatically. At first, HMBDs were presumed to channel copper to the throat of the pump, but this model has been questioned8.

Gourdon and colleagues' structure2 of LpCopA casts fresh light on this issue. The platform that they have discovered in the protein has positive charges around the putative entry site for copper, providing an ideal docking surface for a negatively charged copper donor. But is the donor a copper chaperone, the HMBD or both? This is a difficult question to answer for LpCopA, in part because the mobility of the HMBD prevented the authors from obtaining a fully resolved structure of this region. Nonetheless, the discovery of the platform in LpCopA should enable the identification of platform regions in other P1B-type ATPases for which the structures of isolated HMBDs and copper chaperones are already known, and this should, in turn, allow the question to be answered. Meanwhile, the mobility of the HMBD suggests that it is well suited to regulating the activity of LpCopA — by, for example, forming interactions with the nearby actuator domain of the protein (Fig. 1b).

LpCopA spans the inner cell membrane of L. pneumophila, and pumps copper from the cytosol to the periplasm (the region between the inner and outer membranes; Fig. 1). The protein undergoes cycles of conformational changes that drive the passage of the metal. Gourdon and colleagues' structure2 supplies crucial evidence for how this might occur. The authors observe that three amino-acid residues on the platform of LpCopA are sufficiently close together to simultaneously bind copper ions. They suggest that two of these residues pull apart from the third as the protein shifts into the next conformational state of the pumping cycle. At the same time, other copper-binding amino-acid residues deeper in the membrane approach the third original copper-binding residue. These combined conformational changes should attract copper ions from the platform to binding sites in the transmembrane region of LpCopA. In the final stage of the pumping cycle, the authors propose that the enzyme reverts to its initial state, whereupon a crucial copper-binding residue deep in the transmembrane region is 'tugged' by hydrogen bonds, allowing copper ions to be released to a binding site close to the outer face of the membrane. This site binds the metal only weakly, thus allowing the ion to escape into the periplasm.

P1B-type ATPases that selectively transport the biologically essential metals zinc and cobalt are known, as are others that transport the non-essential metals cadmium and lead. What enables these enzymes to select and pump metals other than copper? One probable answer is that the residues that form metal-binding sites in these proteins — those equivalent to the copper-binding residues in LpCopA — have been replaced with others through evolution, optimizing selective binding to the preferred metal. Indeed, some likely candidates for these alternative metal-binding residues have already been identified by comparison of the proteins' amino-acid sequences with those of copper-pumping enzymes. The charge on the metal-binding platform in these proteins might also repulse, rather than attract, copper chaperones. Alternatively, the HMBDs in the proteins might prevent copper chaperones either from reaching the platforms, or from regulating the activities, of these metal-ion pumps.

Many Gram-negative bacteria have a copper-efflux system that straddles both their inner and outer cell membranes, and exports copper from the cell by means of a ladder of copper-binding residues9. So why do these bacteria also need P1B-type proteins that pump copper only part of the way out of the cell (that is, into the periplasm)? Indeed, why do many bacteria have several different kinds of P1B-type pumps for copper?

One view is that some P1B-type ATPases supply copper as a cofactor to cuproproteins in the periplasm. An alternative view is that some of these proteins pump copper ions from the periplasm into the cytosol — in which case, the conformational changes that generate a unidirectional drive in LpCopA would have to be modified to allow copper ions to flow in the opposite direction. A subset of pumps that have especially high affinities for copper but poor rates of outward transport has been identified, and members of this group are possible candidates for these alternative roles10. Gourdon and colleagues' structure should make it possible to identify parts of the copper-transport pathway that differ in this subset of P1B-type ATPases.

The ATPase ATP7B is found in liver cells, where it loads excess copper into bile ducts for excretion5. Mutations in ATP7B cause Wilson disease, symptoms of which include failure of copper excretion into bile and accumulation of the metal in the liver. The disease is managed by controlling copper levels in the body, for example, by administering the drug penicillamine, which binds to copper ions. Gourdon and colleagues' structure2 of LpCopA should allow the biochemical consequences of different mutations associated with Wilson disease to be defined, possibly providing opportunities to develop personalized metal-management therapies. Furthermore, occipital horn syndrome and distal motor neuropathy are diseases that result from 'milder' mutations in the Menkes ATPase — the mutations cluster at the surface and extracellular face of the ATPase, unlike Menkes-disease mutations, which cluster near core transport functions of the copper pump2. The authors' findings might also provide fresh avenues of investigation to improve treatments for these diseases.