P-type ATPase

The P-type ATPases, also known as E₁-E₂ ATPases, are a large group of evolutionarily related ion and lipid pumps that are found in bacteria, archaea, and eukaryotes. P-type ATPases are α-helical bundle primary transporters named based upon their ability to catalyze auto- phosphorylation of a key conserved aspartate residue within the pump and their energy source, adenosine triphosphate. In addition, they all appear to interconvert between at least two different conformations, denoted by E₁ and E₂. P-type ATPases fall under the P-type ATPase Superfamily which, as of early 2016, includes 20 different protein families.
Most members of this transporter superfamily drive cation uptake or efflux, though one subfamily, the flippases, is involved in flipping phospholipids to maintain the asymmetric nature of the biomembrane.
In humans, P-type ATPases serve as a basis for nerve impulses, relaxation of muscles, secretion and absorption in the kidney, absorption of nutrient in the intestine and other physiological processes. Prominent examples of P-type ATPases are the sodium-potassium pump, the proton-potassium pump, the calcium pump and the plasma membrane proton pump of plants and fungi.

General transport reaction

The generalized reaction for P-type ATPases is
nLigand₁ + mLigand₂ + ATP → nLigand₁ + mLigand₂ + ADP + P_i.
where the ligand can be either a metal ion or a phospholipid molecule.

Discovery

The first P-type ATPase discovered was the Na⁺/K⁺-ATPase, which Nobel laureate Jens Christian Skou isolated in 1957. The Na⁺/K⁺-ATPase was only the first member of a large and still-growing protein family.

Structure

P-type ATPases have a single catalytic subunit of 70 - 140 kDa. The catalytic subunit hydrolyzes ATP, contains the aspartyl phosphorylation site and binding sites for the transported ligand and catalyzes ion transport. Various subfamilies of P-type ATPases also need additional subunits for proper function. Additional subunits that lack catalytic activity are present in the ATPase complexes of P1A, P2A, P2C and P4 ATPases. E.g. the catalytic alpha subunit of Na⁺/K⁺-ATPase consists of two additional subunits, beta and gamma, involved in trafficking, folding, and regulation of these pumps. The first P-type ATPase to be crystallized was SERCA1a, a sarcoplasmic reticulum Ca²⁺-ATPase of fast twitch muscle from adult rabbit. It is generally acknowledged that the structure of SERCA1a is representative for the superfamily of P-type ATPases.
The catalytic subunit of P-type ATPases is composed of a cytoplasmic section and a transmembrane section with binding sites for the transported ligand. The cytoplasmic section consists of three cytoplasmic domains, designated the P, N, and A domains, containing over half the mass of the protein.

Membrane section

The transmembrane section typically has ten transmembrane helices, with the binding sites for transported ligand is predicted to have 8 transmembrane helices. P5 ATPases appear to have a total of 12 transmembrane helices.
Common for all P-type ATPases is a core of six transmembrane-spanning segments, that harbors the binding sites for the translocated ligand. The ligand enter through a half-channel to the binding site and leave on the other side of the membrane through another half-channel.
Varying among P-type ATPase is the additional number of transmembrane-spanning segments domain', which between subfamilies ranges from two to six. Extra transmembrane-segments likely provide structural support for the T domain and can also have specialized functions.

Phosphorylation (P) domain

The P domain contains the canonical aspartic acid residue phosphorylated during the reaction cycle. It is composed of two parts widely separated in sequence. These two parts assemble into a seven-strand parallel β-sheet with eight short associated a-helices, forming a Rossmann fold.
The folding pattern and the locations of the critical amino acids for phosphorylation in P-type ATPases has the haloacid dehalogenase fold characteristic of the haloacid dehalogenase superfamily, as predicted by sequence homology. The HAD superfamily functions on the common theme of an aspartate ester formation by an S_N2 reaction mechanism. This S_N2 reaction is clearly observed in the solved structure of SERCA with ADP plus AlF₄⁻.

Nucleotide binding (N) domain

The N domain serves as a built-in protein kinase that functions to phosphorylate the P domain. The N domain is inserted between the two segments of the P domain, and is formed of a seven-strand antiparallel β-sheet between two helix bundles. This domain contains the ATP-binding pocket, pointing out toward the solvent near the P-domain.

Actuator (A) domain

The A domain serves as a built-in protein phosphatase that functions to dephosphorylate the phosphorylated P domain. The A domain is the smallest of the three cytoplasmic domains. It consists of a distorted jellyroll structure and two short helices. It is the actuator domain modulating the occlusion of the transported ligand in the transmembrane binding sites, and it is pivot in transposing the energy from the hydrolysis of ATP in the cytoplasmic domains to the vectorial transport of cations in the transmembrane domain. The A domain dephosphorylates the P domain as part of the reaction cycle using a highly conserved TGES motif located at one end of the jellyroll.

Regulatory (R) domain

Some members of the P-type ATPase family have additional regulatory domains fused to the pump. Heavy metal P1B pumps can have several N- and C-terminal heavy metal-binding domains that have been found to be involved in regulation. The P2B Ca²⁺ ATPases have autoinbitory domains in their amino-terminal or carboxy-terminal regions, which contain binding sites for calmodulin, which, in the presence of Ca²⁺, activates P2B ATPases by neutralizing the terminal constraint. The P3A plasma membrane proton pumps have a C-terminal regulatory domain, which, when unphosphorylated, inhibits pumping.

Mechanism

All P-type ATPases use the energy derived from ATP to drive transport. They form a high-energy aspartyl-phosphoanhydride intermediate in the reaction cycle, and they interconvert between at least two different conformations, denoted by E₁ and E₂. The E₁-E₂ notation stems from the initial studies on this family of enzymes made on the Na⁺/K⁺-ATPase, where the sodium form and the potassium form are referred to as E₁ and E₂, respectively, in the "Post-Albers scheme". The E₁-E₂ schema has been proven to work, but there exist more than two major conformational states. The E₁-E₂ notation highlights the selectivity of the enzyme. In E₁, the pump has high affinity for the exported substrate and low affinity for the imported substrate. In E₂, it has low affinity of the exported substrate and high affinity for the imported substrate. Four major enzyme states form the cornerstones in the reaction cycle. Several additional reaction intermediates occur interposed. These are termed E₁~P, E₂P, E₂-P*, and E₁/E₂.
ATP hydrolysis occurs in the cytoplasmic headpiece at the interface between domain N and P. Two Mg-ion sites form part of the active site. ATP hydrolysis is tightly coupled to translocation of the transported ligand through the membrane, more than 40 Å away, by the A domain.

Classification

A phylogenetic analysis of 159 sequences made in 1998 by Axelsen and Palmgren suggested that P-type ATPases can be divided into five subfamilies, based strictly on a conserved sequence kernel excluding the highly variable N and C terminal regions. Chan et al. also analyzed P-type ATPases in all major prokaryotic phyla for which complete genome sequence data were available and compared the results with those for eukaryotic P-type ATPases. The phylogenetic analysis grouped the proteins independent of the organism from which they are isolated and showed that the diversification of the P-type ATPase family occurred prior to the separation of eubacteria, archaea, and eucaryota. This underlines the significance of this protein family for cell survival under stress conditions.

P1 ATPases

P1 ATPases consists of the transition/heavy metal ATPases. Topological type I P-type ATPases predominate in prokaryotes.

P1A ATPases (potassium pumps)

P1A ATPases are involved in K⁺ import. They are atypical P-type ATPases because, unlike other P-type ATPases, they function as part of a heterotetrameric complex, where the actual K⁺ transport is mediated by another subcomponent of the complex.

P1B ATPases (heavy metal pumps)

P1B ATPases are involved in transport of the soft Lewis acids: Cu⁺, Ag⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺ and Co²⁺. They are key elements for metal resistance and metal homeostasis in a wide range of organisms.
Metal binding to transmembrane metal-binding sites in Cu⁺-ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu⁺ does not access Cu⁺-ATPases in a free form but is bound to a chaperone protein. The delivery of Cu⁺ by Archaeoglobus fulgidus Cu⁺-chaperone, CopZ, to the corresponding Cu⁺-ATPase, CopA, has been studied. CopZ interacted with and delivered the metal to the N-terminal metal binding domain of CopA. 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 altogether. Thus, MBDs may serve a regulatory function without participating directly in metal transport, and the chaperone delivers Cu⁺ directly to transmembrane transport sites of Cu⁺-ATPases. Wu et al. have determined structures of two constructs of the Cu pump from Archaeoglobus fulgidus by cryoelectron microscopy of tubular crystals, which revealed the overall architecture and domain organization of the molecule. They localized its N-terminal MBD within the cytoplasmic domains that use ATP hydrolysis to drive the transport cycle and built a pseudoatomic model by fitting existing crystallographic structures into the cryoelectron microscopy maps for CopA. The results also similarly suggested a Cu-dependent regulatory role for the MBD.
In the Archaeoglobus fulgidus CopA, invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites. 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 milieu. Transport studies have shown that most 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. This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments. Structural and mechanistic details of copper-transporting P-type ATPase functionhave been described.