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The P-type ATPases, also known as E1-E2 ATPases, are a large group of evolutionarily related ion pump and lipid pumps that are found in bacteria, archaea, and eukaryotes. P-type are α-helical bundle active transport named based upon their ability to catalyze auto- (or self-) phosphorylation (hence P) of a key conserved aspartate residue within the pump and their energy source, adenosine triphosphate (ATP). 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 (P-ATPase) Superfamily ( TC# 3.A.3) which, as of early 2016, includes 20 different protein families.
Most members of this transporter superfamily catalyze cation uptake and/or efflux, however one subfamily, the , ( TC# 3.A.3.8) is involved in flipping phospholipids to maintain the asymmetric nature of the biomembrane.
In humans, P-type ATPases serve as a basis for Action potential, relaxation of muscles, Renal function, absorption of nutrient in the intestine and other physiological processes. Prominent examples of P-type ATPases are the sodium-potassium pump (Na+/K+-ATPase), the proton-potassium pump (H+/K+-ATPase), the SERCA (Ca2+-ATPase) and the plasma membrane proton pump (H+-ATPase) of plants and fungi.
nLigand1 (out) + mLigand2 (in) + ATP → nLigand1 (in) + mLigand2 (out) + ADP + Pi.
where the ligand can be either a metal ion or a phospholipid molecule.
The catalytic subunit of P-type ATPases is composed of a cytoplasmic section and a transmembrane section with binding sites for the transported ligand(s). The cytoplasmic section consists of three cytoplasmic domains, designated the P, N, and A domains, containing over half the mass of the protein.
Common for all P-type ATPases is a core of 6 transmembrane-spanning segments (also called the 'transport (T) domain'; M1-M6 in SERCA), that harbors the binding sites for the translocated ligand(s). The ligand(s) 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 (also called the 'support (S) domain', which between subfamilies ranges from 2 to 6. Extra transmembrane-segments likely provides structural support for the T domain and can also have specialized functions.
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 (HAD) superfamily, as predicted by sequence homology. The HAD superfamily functions on the common theme of an aspartate ester formation by an SN2 reaction mechanism. This SN2 reaction is clearly observed in the solved structure of SERCA with ADP plus AlF4−.;
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(s) through the membrane, more than 40 Å away, by the A domain.
Metal binding to transmembrane metal-binding sites (TM-MBS) 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 (see TC# 3.A.3.5.7), to the corresponding Cu+-ATPase, CopA ( TC# 3.A.3.5.30), has been studied. CopZ interacted with and delivered the metal to the N-terminal metal binding domain(s) of CopA (MBDs). 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. (2008) have determined structures of two constructs of the Cu (CopA) 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 ( TC# 3.A.3.5.7), invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). 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 (or alternative functions). 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.
Crystal structures of Sarcoplasimc/endoplasmic reticulum ATP driven calcium pumps can be found in RCSB.
SERCA is composed of a cytoplasmic section and a transmembrane section with two Ca2+-binding sites. The cytoplasmic section consists of three cytoplasmic domains, designated the P, N, and A domains, containing over half the mass of the protein. The transmembrane section has ten transmembrane helices (M1-M10), with the two Ca2+-binding sites located near the midpoint of the bilayer. The binding sites are formed by side-chains and backbone carbonyls from M4, M5, M6, and M8. M4 is unwound in this region due to a conserved proline (P308). This unwinding of M4 is recognised as a key structural feature of P-type ATPases.
Structures are available for both the E1 and E2 states of the Calcium ATPase showing that Ca2+ binding induces major changes in all three cytoplasmic domains relative to each other.
In the case of SERCA, energy from ATP is used to transport 2 Ca2+-ions from the cytoplasmic side to the lumen of the sarcoplasmatic reticulum, and to countertransport 1-3 protons into the cytoplasm. Starting in the E1/E2 state, the reaction cycle begins as the enzyme releases 1-3 protons from the cation-ligating residues, in exchange for cytoplasmic Ca2+-ions. This leads to assembly of the phosphorylation site between the ATP-bound N domain and the P domain, while the A domain directs the occlusion of the bound Ca2+. In this occluded state, the Ca2+ ions are buried in a proteinaceous environment with no access to either side of the membrane. The Ca2E1~P state becomes formed through a kinase reaction, where the P domain becomes phosphorylated, producing ADP. The cleavage of the β-phosphodiester bond releases the gamma-phosphate from ADP and unleashes the N domain from the P domain.
This then allows the A domain to rotate toward the phosphorylation site, making a firm association with both the P and the N domains. This movement of the A domain exerts a downward push on M3-M4 and a drag on M1-M2, forcing the pump to open at the luminal side and forming the E2P state. During this transition, the transmembrane Ca2+-binding residues are forced apart, destroying the high-affinity binding site. This is in agreement with the general model form substrate translocation, showing that energy in primary transport is not used to bind the substrate but to release it again from the buried counter ions. At the same time the N domain becomes exposed to the cytosol, ready for ATP exchange at the nucleotide-binding site.
As the Ca2+ dissociate to the luminal side, the cation binding sites are neutralised by proton binding, which makes a closure of the transmembrane segments favourable. This closure is coupled to a downward rotation of the A domain and a movement of the P domain, which then leads to the E2-P* occluded state. Meanwhile, the N domain exchanges ADP for ATP.
The P domain is dephosphorylated by the A domain, and the cycle completes when the phosphate is released from the enzyme, stimulated by the newly bound ATP, while a cytoplasmic pathway opens to exchange the protons for two new Ca2+ ions.
Xu et al. proposed how Ca2+ binding induces conformational changes in TMS 4 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, suggested a comparable 70º rotation of N relative to P to deliver ATP to the phosphorylation site.
One report suggests that this sarcoplasmic reticulum (SR) Ca2+ ATPase is homodimeric.
Crystal structures have shown that the conserved TGES loop of the Ca2+-ATPase is isolated in the Ca2 E1 state but becomes inserted in the catalytic site in E2 states. Anthonisen et al. (2006) characterized the kinetics of the partial reaction steps of the transport cycle and the binding of the phosphoryl analogs BeF, AlF, MgF, and vanadate in mutants with alterations to conserved TGES loop residues. The data provide functional evidence supporting a role of Glu183 in activating the water molecule involved in the E2P → E2 dephosphorylation and suggest a direct participation of the side chains of the TGES loop in the control and facilitation of the insertion of the loop in the catalytic site. The interactions of the TGES loop furthermore seem to facilitate its disengagement from the catalytic site during the E2 → Ca2 E1 transition.
Crystal Structures of Calcium ATPase are available in RCSB and include: , , , , among others.
The X-ray crystal structure at 3.5 Å resolution of the pig renal Na+/K+-ATPase has been determined with two rubidium ions bound in an occluded state in the transmembrane part of the α-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+/K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of the sarco(endo)plasmic reticulum. The C-terminus of the α-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.
Crystal Structures are available in RCSB and include: , , , , among others.
Plasma membrane H+-ATPase is best characterized in plants and yeast. It maintains the level of intracellular pH and transmembrane potential. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.
In eukaryotes, they are present in the plasma membranes or endoplasmic reticular membranes. In prokaryotes, they are localized to the cytoplasmic membranes.
P-type ATPases from 26 eukaryotic species were analyzed later.
Chan et al., (2010) conducted an equivalent but more extensive analysis of the P-type ATPase Superfamily in Prokaryotes and compared them with those from Eukaryotes. While some families are represented in both types of organisms, others are found only in one of the other type. The primary functions of prokaryotic P-type ATPases appear to be protection from environmental stress conditions. Only about half of the P-type ATPase families are functionally characterized.
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