Human Coppertransporting Atpases As Members Of The Ptype Atpase Family

Analysis of the primary sequence of the ATP7A and ATP7B gene products revealed that the corresponding proteins, MNKP and WNDP, belong to a family of cation-transporting P-type ATPases. The P-type ATPases are a large group of membrane proteins that utilize energy of ATP hydrolysis to transport various ions across cell membranes. During the catalytic cycle the y-phosphate of ATP is transferred to the invariant Asp residue within the nucleotide-binding site of ATPase with the formation of acylphosphate intermediate; this property distinguish the P-type ATPases from other cation-transporting pumps. Because both Wilson's disease and Menkes disease are associated with defects in copper distribution, it was proposed that WNDP and MNKP function in a cell as copper-transporting P-type ATPases. In agreement with this proposal, recent studies by Voskoboinik et al. have shown that MNKP and WNDP transport copper across cell membranes and ATP stimulates the MNKP- and WNDP-dependent transport (19,20).

Today, over 100 members of the P-type ATPase family has been described and a wealth of information has been accumulated regarding the structure and function of some of these proteins (for review, see refs. 21 and 22). The first crystal structure of a P-type ATPase, the Ca2+-ATPase from sarcoplasmic reticulum, has been recently solved, providing an important framework for studies on molecular mechanisms of the ATP-driven ion transport (23). To understand how this information can be utilized for analysis of WNDP and MNKP, it could be beneficial to dissect features that are common for the human copper-transporting ATPases and other P-type ATPases, as well as to identify the unique structural and functional characteristics of MNKP and WNDP.

Like all P-type ATPases, WNDP and MNKP have several highly conserved sequence motifs, such as DKTG, TGDN, GDGxxD, and TGEA/S (Fig. 1). The invariant residues in these motifs are known to play key roles in catalysis and accompanying conformational transitions, indicating that the basic mechanisms of ATP hydrolysis and coupling between the hydrolytic and ion-transport steps are likely to be the same for human copper-transporting ATPases and other P-type pumps.

At the same time, human copper-transporting ATPases have several unique structural and functional characteristics (see Sections 2.2. and 2.4.), indicating that specific details of their molecular mechanism and their intracellular behavior differ from those of well-characterized P-type ATPases. For this reason, in our early attempt to classify the P-type ATPases, we placed the copper-transporting and other structurally similar transient metal-transporting ATPases into a separate subgroup (P1-type ATPases), in contrast to P2-ATPases, such as Ca2+-ATPase and Na+,K+-ATPase, which transport alkali and alkali-earth ions (24). Solioz and Vulpe later suggested an alternative name for the P1-ATPases, CPx-ATPases, based on the presence of characteristic motif CPx in the transmembrane portion of these proteins (25). In a recent and more complete classification scheme, Axelson and Palmgren also placed the P1-ATPases in a separate group (type IB), distinct from four other P-type ATPase subfamilies (26).

The comparison of structural and functional properties of mammalian copper-transporting AT-Pases (P1-ATPases) and P2-type ATPases reveals the following interesting differences between these two groups of pumps.

2.1. Physiological Role

The major function of all well-characterized P2-type ATPases is to maintain the concentration gradient of the transported cations across cell membranes; the generated gradients then serve as a driving force for such physiological processes as muscle contraction, nutrients uptake, or electrical activity of neurons. Whether human copper-ATPases have a similar role and maintain a concentration gradient of copper across cell membranes remains to be elucidated. In the cytosol, essentially all copper apparently exists in a protein-bound form (27), but its status in the intracellular compartments is less clear. If, in organelles, copper is present in a free form and therefore the transmembrane copper gradient is generated, it remains unknown whether such gradient drives any secondary process. However, it is now well established that the eucaryotic copper-transporting ATPases represent key components of a biosynthetic, cofactor-delivery pathway, transporting copper to copper-dependent enzymes. Incorporation of copper into proteins in the secretory pathway is essential for numerous physiological functions, including respiration, neurotransmitter biosynthesis, and high-affinity iron uptake; however, the role of copper in these processes is indirect.

In addition to their important role in delivering copper to the copper-dependent enzymes, human copper-transporting ATPases regulate the intracellular concentration of copper by removing excess copper from the cell. This "detoxification" function of copper-transporting ATPases is very similar to the functional role of bacterial Cd2+-ATPase and Pb2+-ATPases and likely appeared first during evolution. It was later extended to accommodate eucaryotic cell needs in having copper inside various cell organelles. To carry out this dual function, WNDP and MNKP have to be located, at least temporarily, in different cell compartments.

In agreement with this prediction, MNKP was shown to cycle between the trans-Golgi network (TGN) and the plasma membrane: Under basal conditions, MNKP was detected predominantly in TGN, whereas increase in copper concentration led to the redistribution of MNKP from TGN to the plasma membrane (28,29). Similarly, the intracellular localization of WNDP depends on copper

Fig. 2. Comparison of the transmembrane topology and organization of the cation-translocation pathway in human copper-transporting ATPases (P1-type ATPases) and P2-type ATPases. The letters and the arrows/vertical lines mark the positions of the conserved sequence motifs; Pi indicates the Asp residue in the DKTG motif that is phosphorylated during the catalytic cycle and the asterisk shows the position of the TGEA/S sequence in the transmembrane model of ATPases. The transmembrane segments that are common for the P1- and P2-ATPases are dark colored, the membrane segments unique for each group of ATPases are light gray; the open circles indicate the transmembrane segments known to be important for cation coordination and transport activity in both groups of pumps.

Fig. 2. Comparison of the transmembrane topology and organization of the cation-translocation pathway in human copper-transporting ATPases (P1-type ATPases) and P2-type ATPases. The letters and the arrows/vertical lines mark the positions of the conserved sequence motifs; Pi indicates the Asp residue in the DKTG motif that is phosphorylated during the catalytic cycle and the asterisk shows the position of the TGEA/S sequence in the transmembrane model of ATPases. The transmembrane segments that are common for the P1- and P2-ATPases are dark colored, the membrane segments unique for each group of ATPases are light gray; the open circles indicate the transmembrane segments known to be important for cation coordination and transport activity in both groups of pumps.

concentration (30,31). In response to increased copper, WNDP redistributes from its primary localization site, TGN, to a vesicular compartment (probably endosomes). Thus, changes in copper concentration seem to regulate copper transport across various cell membranes by altering the number of copper transporters present at these membranes. So far, the dependence of intracellular localization on concentration of the transported ion seems to be a unique property of human copper-transporting ATPases. Whether this mode of regulation is the only way of altering copper transport across the membranes or whether the changes in copper concentrations also control the activity of MNKP and WNDP remains to be elucidated.

2.2. Transport Characteristics

Copper can bind to proteins in either reduced, Cu1+, or in the oxidized, Cu2+, form. The ability of copper to exist in different oxidation states raises an interesting possibility that copper oxidation may occur in the secretory pathway or in another intracellular organelle as the last step of the copper-transport process. Copper binds to WNDP and MNKP in the reduced form (see Section 3.) and is likely to be transported in the same form (32). Copper is then released from the transporters, possibly with a change in oxidation state, and becomes incorporated into copper-dependent enzymes. [Inter estingly, chloride ions seems to play an important role in this process, at least in yeast (33)]. The alternative possibility is that copper is picked up from the ATPases through direct intermolecule interactions either by target proteins or by low-molecular-weight copper carriers. If this last scenario is correct, then the intracellular transport of copper is unique, because the entire ion-transport process would be mediated through a chain of specific protein-protein interactions.

2.3. Cation Recognition

The extremely low concentrations of free copper in a cell (27) results in another interesting property of WNDP and MNKP. Unlike many P-type ATPases, which recognize free cations present in the cytosol, eucaryotic copper-transporting ATPases receive copper from so-called copper chaperones, small cytosolic proteins that presumably work as shuttles between the copper uptake system and other components of the copper distribution pathway (34-36). Thus, specificity of MNKP and WNDP for the transported ion is defined not only by the stereochemistry of copper binding sites, but also by specific recognition of the copper-carrier protein, HAH1.

2.4. The Structural Differences Between the P1- and P2-ATPases

It is probably not a coincidence that in addition to the functional differences described earlier, copper-transporting ATPases have structural features that make them quite distinct from the P2-AT-Pases. The most obvious difference is the organization of the cation-translocation pathway (Fig. 2). In their membrane portion, P2-ATPases have 10 transmembrane segments: 4 segments before the ATP-binding domain and 6 segments in the C-terminal portion after the ATP-binding domain. The transmembrane segments involved in cation coordination and transport in the P2-ATPases contain a large number of hydrophilic and helix-breaking amino acid residues, which are essential for binding of the positively charged ions in the membrane (37-39).

In contrast, the P1-ATPases have a total of eight membrane-spanning regions: six before the ATP-binding domain, and only one pair after the ATP-binding domain. The membrane portion of the P1-ATPases has fewer hydrophilic and helix-breaking residues, and the segments, corresponding to the last four C-terminal transmembrane helixes of P2-ATPases, are absent in the structure of copper pumps (Fig. 2). Interestingly, these last four transmembrane segments play an important role in the insertion and maintenance of the ion-binding segments in P2-ATPases and may even contribute to cation coordination (40,41). The absence of these fragments in the copper-ATPases suggests that the P1- and P2-ATPases likely to have different mechanisms for insertion of some transmembrane hairpins and for overall assembly of the cation-translocation pathway.

It is also interesting that the transmembrane segments immediately after the ATP-binding domain, which play a central role in ion coordination in the P2-type ATPases, do not contain any obvious ligands for copper binding in WNDP and MNKP. Currently, the only candidate for copper binding in the membrane is the CPC motif in the sixth transmembrane helix (see Fig. 1). This suggests that additional coordination of copper could be provided either by side chains of Ser/Thr and Tyr residues or by the backbone carbonyls of other transmembrane segments. Alternatively, it is possible that two additional transmembrane segments present at the N-terminal part of the protein are required to form the copper-translocation pathway. If the first membrane hairpin, which is absent in the structure of the P2-ATPases, is directly involved in copper transport, then the mechanism of coupling between the ATP hydrolysis and cation transport could be quite different for the P1-type and P2-type pumps.

Another obvious difference between the P1- and P2-type ATPases is the role of the N-terminal domain in cation binding and selectivity. Mutations in the N-terminal domain do not have a dramatic effect on the cation affinity of the characterized P2-ATPases, which is defined mainly by the residues located in the transmembrane portion of these proteins ([22,42] and Fig. 2). What specific role the N-terminus of the P2-ATPases plays in the transport process, if any, is still not clear. In contrast, the N-terminal domain of the copper-transporting ATPases contains multiple copper-binding sites (see Fig.

Fig. 3. (A) Expression of N-WNDP-HT in Escherichia coli and purified soluble protein used for further analysis of the secondary structure. (B) Circular dichroism spectroscopy of N-WND-HT reveals the following secondary structure elements for this domain: 19.5% a-helix, 26% p-sheet, 21% p-turn, and 33.5% random coil.

Fig. 3. (A) Expression of N-WNDP-HT in Escherichia coli and purified soluble protein used for further analysis of the secondary structure. (B) Circular dichroism spectroscopy of N-WND-HT reveals the following secondary structure elements for this domain: 19.5% a-helix, 26% p-sheet, 21% p-turn, and 33.5% random coil.

1 and text below), and the presence of at least one or more of these repeats is essential for copper transport by MNKP and WNDP (34,43-45). This interesting difference between the P1- and P2-AT-Pases seemed critical for dissecting the molecular mechanism of copper transport and prompted us and other investigators to focus our attention on biochemical characterization of the N-terminal domain of WNDP and MNKP.

0 0

Post a comment