Sandbox Reserved 811

This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.

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Na+,K+ ATPaseNa+,K+ ATPase

spinqualitylabelsall models Crystal structure of the sodium-potassium pump Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The Na+,K+-ATPase is a transmembrane protein which generates an electrochemical gradient for sodium and potassium ions thanks to the hydrolysis of ATP. This pump exchanges 3Na+ for 2K+ (out) consuming 1ATP. It belongs to the family of P-type ATPase, also known as E1-E2 ATPases, which are phosphorylated on an Aspartateresidue during the transport cycle. It is vital to animal cells, to regulate their volume and to keep their homeostasis. Depending on the cells where the pump is, it allows the formation of a membrane potential or the transport of molecules through the membrane due to the created ionic concentration gradients. These gradients provide energy for other secondary active transports.

StructreStructre

The pump has a total length of about 16.5 to 18.5 nm. The extensions of the protein are 5 nm long beyond the cytoplasmic surface and 1 to 3 nm long beyond the extracellular surface. Its mass is asymmetrically distributed across the membrane: it is 3 to 4 times more important on the cytoplasmic side. The mass of the α-subunit exposed on the extracellular surface is 3-4 times more important than on the cytoplasmic one. The catalytic functions are executed in the cytoplasmic domains separated by intramembranous domain. Most of the mass of the β-subunit (whose carbohydrate) are located at the extracellular domain. Experiments showed that there is a distance of 7-8 nm between the binding sites for ATP and for K+.

Tryptophan residues, sulfhydryl groups, ionisable groups and intramembranous segments are involved in the structural conformation of the α-subunit. There is a transition from E1 to E2 conformation: it is a change of secondary structure (α-helix into β-sheet transition) involving at least 80 amino acids.

α-subunitα-subunit

The α-subunit has three highly-conserved domains: the actuator domain (A), the nucleotide-binding domain (N) and the phosphorylation domain (P). Their conformation changes in response to ligand binding that allows ion exchange across the membrane. These domains are along the Na+ and K+ binding sites. Indeed, two sites, which bind 2K+ or 2Na+, locate between helices 4, 5 and 6. The third Na+ is bound on the carboxyl-terminal domain. The α-subunit is a 1021 residues and 112 kDa polypeptide. It is the catalytic component of the enzyme. It has binding domains for ATP and the cations. So it catalyzes the hydrolysis of ATP into ADP and permits the exchange of Na+ and K+. It owns a transmembrane domain made of 10 alpha-helix, αM1 to αM10. αM4 and αM6 are partially unwound to form a pocket for K+ ions. K+ binding sites, called 1 and 2, are located between αM4, αM5 and αM6. Several residues are involved in cations binding and their stabilization: Asp-804 donates a side-chain oxygen ligand to sites 1 and 2, Glu-327 to site 2. The side-chain of Ser-775, Asn-776 and Glu-779 may, directly or by a molecule of water, donate ligands for binding. There is a potentially intervention of the residues Asp-808 and Gln-923, which are situated further away. Side chains of Glu 327, Ser 775, Asn 776, Glu 779 and Asp 804 are close enough to the sites to bind the ions, either directly or via a water molecule. Glu 327 associates only with the site 2 and possibly controls the extracellular gate of occlusion cavity, it might be controlled by contact with the Leu 97 residue. It seems that post-translational modifications modulate the activity of the enzyme. Indeed the phosphorylation on Tyr-10 modulates pumping activity. The phosphatase 2A (PP2A) dephosphorylates this residue when there is an increase in intracellular Na+. This dephosphorylation allows to increase the catalytic activity. 2Na+ bind at the same sites than K+ ions, which supports the consecutive support model, K+ is being fixed once Na+ released via the same occlusion cavity.The third Na+ binds to a site between the C-terminal domain of theα subunit and several nearby residues. A pocket located between αM7 and αM8 recognizes the two latest residues of the C-terminal domain, two Tyr (1015, 1016). The last one interacts with Lys-766 and Arg-933 in the loop connecting αM8 and αM9. The six precedent residues formanα-helix sited between the α-helix of the β-subunit, αM7 and αM10. Tyr-771, Thr-807 and Glu-954 interact with this binding site too. Because they are far away from the C-terminal domain, it seems that they allow to stabilize the interactions between the helices. Near to the C-terminal domain, there is a region of 6 Arg, (1003, 1004, 1005, 933, 934, 998), they make the region around C-term highly electropositive. In many voltage-dependant channels Arginine clusters are voltage sensors and move responding to membrane depolarization. In the sodium potassium pump this cluster could act as a switch that changes relations of C-term in its binding pocket during depolarization/repolarization influencing the affinity for Na+ ions. This subunit is synthesized by membrane-bound ribosomes and cotranslationally inserted into the membrane.

β-subunitβ-subunit

The β-subunit is a glycoprotein of 35 kDa and made of 302 residues. The hydrophilic part of the β-subunit is exposed only on the outer surface of the cell. It is a glycosylated domain which covers the α-subunit in order to prevent the escape of K+ ions. It is involved in integration and proper orientation of the α-subunit into the membrane. It plays a role in ion occlusion during exchange. It owns a transmembrane segment βMmade of 1 α-helix. It directly contacts with αM7 and αM10 of the α-subunit. Tyr-39, Phe-42 and Tyr-43 interacts with Gly-848 of αM7 and the repeated GXXXG motif is exposed on the other side of the βM-helix. The cytosolic N-term of β subunit continues probably aroud the α subunit, but cannot be modelled. The first residues of the β-domain can establish a contact with the αM7 - αM8 loop aroud the SYGQ motif which seems indispensable for αβ assembly.

γ-subunitγ-subunit

Certain pumps are made of a third subunit, the γ-subunit, which plays a role in the regulation of tissue-specific pumping activity. It owns a transmembrane domainγM made of a approximately 30 amino acidsα-helix, which interacts with the α-subunit. Indeed, Glu-953 of αM9 interacts with Gly-41, which is found mutated to arginine in familial dominant renal hypomagnesaemia.Phe-949, Leu-957 and Phe-960 of αM9 interact with other residues of the γ-subunit. The extracellular domainmoves in between the α- and β-subunit and contains a FXYD motif which regulates the activity of the pump by acting on the affinity of the cations for their binding sites.

MechanismMechanism

The transport of these ions is accomplished via a conformational change of the enzyme. Sodium potassium pump is sensitive to the membrane potential, the binding and dissociation of one of the three Na+ ions is voltage-dependent. 3Na+ are bound to the pump in the E1 conformation, ATP is already bound to the α-subunit. It is open to the intracellular milieu of the cell, once 3 Na+ bound it becomes transiently inaccessible to both sides exterior of the plasma membrane. The binding of the sodium ions induces the movement of the A domain: there is occlusion of 2Na+ and hydrolysis of ATP at the P domain. ADP and Na+ bound at the C-terminal domain are released to the extracellular milieu. The pump adopts the E2 conformation and opens to the exterior of the cell. There is an exchange between 2Na+ and 2K+. The α-subunit occludes the potassium ions, inorganic phosphate still bound. Pi is coordinated by loop of Thr-212, Gly-213, Glu-214 and Ser-215. The oxygen of the carboxyl of Glu-213 is H bond distant from MgF¬42-. This conformation is adopted thanks to the surrounding residues, even if it is unstable because of the high electronegativity of F. The enzyme converts to the E1 conformation that induces the replacement of Pi by H2O and the release of K+.