3.A.3 The P-type ATPase (P-ATPase) Superfamily

Nearly all of the members of this superfamily, found in bacteria, archaea and eukaryotes, catalyze cation uptake and/or efflux driven by ATP hydrolysis. Clustering on the phylogenetic tree is usually in accordance with specificity for the transported ion(s). Most of these protein complexes are multisubunit with a large subunit serving the primary ATPase and ion translocation functions. In eukaryotes, they are present in the plasma membranes or endoplasmic reticular membranes. In prokaryotes, they are localized to the cytoplasmic membranes. Gastric H⁺-translocating ATPases comprise a subgroup of the larger and more diverse Na⁺/K⁺ ATPase subfamily (subfamily #1). Ca²⁺ ATPases of eukaryotes comprise a very diverse subfamily (subfamily #2) including both plasma membrane and sarcoplasmic reticular types. Sarcoplasmic reticular Ca²⁺ ATPases (SERCA) in brown adipose tissue can uncouple ATP hydrolysis from Ca²⁺ transport and be thermogenic (de Meis, 2003). H⁺-translocating P-type ATPases of plants and fungi comprise their own subfamily (subfamily #3). Distinct bacterial enzymes specific for K⁺ or Mg²⁺ (uptake), Ca²⁺, Ag⁺, Zn²⁺, Co²⁺, Pb²⁺, Ni²⁺, and/or Cd²⁺ (efflux) and Cu²⁺ or Cu⁺ (uptake or efflux, depending on the system) have been characterized, and each of these enzymes comprises a distinct subfamily. Cu²⁺ or Cu⁺-translocating ATPases from bacteria, archaea and animals cluster together, and at least some of these also transport Ag⁺. The Cu⁺/Ag⁺ ATPases have an 8 TMS topology (Mandal et al., 2002). A cys-pro-cys motif in CopA of E. coli (TC #3.A.3.5.5) is essential for Cu⁺/Ag⁺ efflux and phosphoenzyme formation (Fan and Rosen, 2002).

Many eukaryotic P-type ATPases are homodimers of the catalytic subunit that hydrolyzes ATP, contains the aspartyl phosphorylation site and catalyzes ion transport. The Na⁺,K⁺-ATPases, the Ca²⁺-ATPases and the (fungal) H⁺-ATPases of higher organisms exhibit 10 transmembrane α-helical spanners (TMSs), some of them highly tilted. An S. aureus plasmid p1258 Cd²⁺ ATPase (CadA) has an 8 TMS topology (Tsai et al., 2002). However, additional subunits that appear to lack catalytic activity may be present in the ATPase complex. For example, the 10 TMS catalytic α-subunit of the Na⁺,K⁺-ATPase of animals is tightly complexed to the 1 TMS β-subunit and the tissue-specific, regulatory, 1 TMS γ-subunit. The β-subunit, which may influence the activity of the α-subunit, probably functions to facilitate proper insertion of the α-subunit into the membrane, to allow proper targeting to a subcellular membrane site in post-translational processing, and to stabilize the catalytic subunit. The β-subunit can therefore be considered to be an auxiliary protein of the Na⁺,K⁺-ATPase catalytic subunit. The γ-subunit of the Na⁺,K⁺-ATPase has been reported to influence kinetic parameters and is homologous to a family of pore-forming peptides, the peptides of the phospholemman family (TC #1.A.27). The Na⁺, K⁺-ATPase can serve as a steroid hormone receptor (Schoner, 2002). Several other P-type ATPases also depend on small proteolipids, the functions of which are uncertain.

Considerable evidence is available showing that animals have a Cl^- translocating, Cl^- stimulating P-type ATPase. Although extensive biochemical data are available, the protein sequence of any one such Cl^- ATPase has not yet been determined (Gerencser, 1993; Inagaki et al., 1996; Zeng et al., 1999). Evidence for mammalian iron-inducible, iron-transporting ATPases is also available (Baranano et al., 2000). Finally bacterial Na⁺-transporting P-type ATPases probably exist (Ueno et al., 2000). Thus the breadth of substrates transported by P-type ATPases is likely to be much greater than currently recognized.

The stoichiometries of transport are sometimes known and complex. In the case of the Na⁺,K⁺-ATPases, 3 Na⁺ are exchanged for 2 K⁺ per ATP molecule hydrolyzed. The gastric H⁺-translocating ATPases replace H⁺ for K⁺ but with an H⁺/K⁺ stoichiometry of 2:2. Thus, although these two enzymes are ~65% identical, the Na⁺,K⁺-ATPases are electrogenic while the H⁺,K⁺-ATPases are electroneutral. The Ca²⁺ ATPases may catalyze Ca²⁺-K⁺ antiport. A single organism may possess multiple isoforms of these enzymes. Some members of the P-type ATPase family have been reported to flip phospholipids from one monolayer of the bilayer membrane to the other monolayer.

The structure of the sarcoplasmic reticular Ca²⁺-ATPase has been solved at 2.6 Å resolution for the complex to which 2 Ca²⁺ are bound, and at 3.1 Å resolution for the complex lacking Ca²⁺ (Toyoshima et al., 2000; Toyoshima and Nomura, 2002). The two Ca²⁺ are located side by side, surrounded by 4 transmembrane helices, two of which are unwound for efficient coordination geometry. There are 3 cytoplasmic domains, one the central catalytic domain bearing the phosphorylation site, a second bearing the adenosine nucleotide binding site, and a third of unknown function. The central domain has the same fold as haloacid dehydrogenases (Aravind et al., 1998; Stokes and Green, 2000). The Ca²⁺-free form shows large conformational differences from the Ca²⁺-bound form with the three cytoplasmic domains tightly associated to form a single headpiece and six of the ten TMSs largely rearranged. These latter rearrangements guarantee the release of external Ca²⁺ and create a pathway for entry of Ca²⁺ from the cytoplasm. ATPase activity and Ca²⁺ binding are cooperatively interdependent, but the two processes can be separated by mutations (Zhang et al., 2002).

Structures are available for both the E1 and E2 states of the Ca²⁺ ATPase showing that Ca²⁺ binding induces major changes in all three cytoplasmic domains relative to each other (Xu et al., 2002). Xu et al. propose how Ca²⁺ binding induces conformational changes in TMS4 and 5 in the membrane domain (M) that in turn induce rotation of the phosphorylation domain (P). The nucleotide binding (N) and β-sheet (β) domains are highly mobile, with N flexibly linked to P, and β flexibly linked to M. Modeling of the fungal H⁺ ATPase, based on the structures of the Ca²⁺ pump, suggests a comparable 70º rotation of N relative to P to deliver ATP to the phosphorylation site (Kühlbrandt et al., 2002). The probable H⁺ pathway through the membrane is also revealed.

The Na⁺,K⁺-ATPase can be transformed into an ion channel using pharmacological agents. Palytoxin (PTX) produced by soft coral of the genus Palythoa, binds to the ATPase with a Kd of 20 pM and creates a monovalent cation-selective channel with a single channel conductance of 10 pS. The presence of external Na⁺ seems to be essential for channel activation (Wu et al., 2003). When the N-terminal 35 residues are removed from the ATPase, the toxin-activated channel does not exhibit a time-dependent inactivation gating at positive potentials as is characteristic of the wild-type protein. The truncated pump exhibits no electrogenic current, and the ion stoichiometry for active transport is altered. Addition of the synthetic peptide restores activity towards wild type. The N-terminal peptide therefore appears to act as an inactivation gate (similar to Shaker B channels of the VIC family (TC #1.A.1)). It may also play a critical role in determining the ion stoichiometry (Wu et al., 2003).

The generalized reaction for P-type ATPases is:

nMe₁ (out) + mMe₂ (in) + ATP → nMe₁ (in) + mMe₂ (out) + ADP + P_i.

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Examples: