Structural basis for gating mechanism of the human sodium-potassium pump

Abstract

P2-type ATPase sodium-potassium pumps (Na+/K+-ATPases) are ion-transporting enzymes that use ATP to transport Na+ and K+ on opposite sides of the lipid bilayer against their electrochemical gradients to maintain ion concentration gradients across the membranes in all animal cells. Despite the available molecular architecture of the Na+/K+-ATPases, a complete molecular mechanism by which the Na+ and K+ ions access into and are released from the pump remains unknown. Here we report five cryo-electron microscopy (cryo-EM) structures of the human alpha3 Na+/K+-ATPase in its cytoplasmic side-open (E1), ATP-bound cytoplasmic side-open (E1•ATP), ADP-AlF4− trapped Na+-occluded (E1•P-ADP), BeF3− trapped exoplasmic side-open (E2P) and MgF42− trapped K+-occluded (E2•Pi) states. Our work reveals the atomically resolved structural detail of the cytoplasmic gating mechanism of the Na+/K+-ATPase.

Solid-state NMR chemical shift analysis for determining the conformation of ATP bound to Na,K-ATPase in its native membrane

“…Crystal structures have been determined for NKA in the E1·Na + state in the presence of the ADP·AlF 4 − proxy to trap the enzyme in the transition state preceding E1P (PDB and ; resolution 4.3 Å and 2.8 Å), 40,41 and in the E1 state with the AMPPCP proxy (PDB ; 3.5 Å). 42 Inspection of the structures of ATP and its analogues in these binding sites indicates that the ribose group adopts N-type conformations in and (Fig. 1b and c, left) and S-type conformations in and (Fig.…”

“…Existing atomic structures of NKA indicate that there is no clear relationship between the ATP ribose conformation and the conformation adopted by NKA in the catalytic cycle required for ion pumping. [38][39][40][41][42] Proteins that use ATP for energy source tend to bind ATP with a C2 ′ -endo ribose, whereas proteins that use ATP for phosphorylation tend to bind ATP with a C3 ′ -endo ribose. 51 NKA utilises the energy of ATP hydrolysis and is also phosphorylated, so it is unsurprising that both ribose conformations are adopted.…”

“…The white crosses denote the ribose conformations of ATP in: the cryo-EM structure of NKA in the E2P state formed in the presence of ATP (PDB 7WYU); 39 the E2.2K + state after addition of ATP (PDB 7Y46); 38 the E1$Na + state with ADP and AlF 4 − (PDB 3WGU); 41 and the E1 state with AMPPCP (PDB 8D3W). 42 (c) Ribose ring conformations of ATP, AMPPCP and ADP in the 4 NKA structures highlighted in (b). (d) Plane wave-GIPAW DFT calculations (with the GGA functional) of 13 C isotropic chemical shifts for ATP ribose carbons.…”

“…(b) Distribution of ATP ribose ring conformations in 272 PDB files of protein structures.Conformations are represented by pseudorotation factor P and maximum amplitude q max . The white crosses denote the ribose conformations of ATP in: the cryo-EM structure of NKA in the E2P state formed in the presence of ATP (PDB 7WYU); 39 the E2.2K + state after addition of ATP (PDB 7Y46); 38 the E1$Na + state with ADP and AlF 4 − (PDB 3WGU);41 and the E1 state with AMPPCP (PDB 8D3W) 42. (c) Ribose ring conformations of ATP, AMPPCP and ADP in the 4 NKA structures highlighted in (b).…”

ATP1A3 Disease Spectrum Includes Paroxysmal Weakness and Encephalopathy Not Triggered by Fever

“… 33 Val129-Val130 lie within the transmembrane ion-binding domain in transmembrane helix M2, which interacts with helix M1, runs alongside 2 helices (M4 and M6) that form the ion-binding sites ( Figure 1 ), and rotates relative to the latter during each catalytic cycle. 24 , 34 Deletion of one valine might displace the rest of the M2 side chains by 100°, disrupting these interactions, or distorting the local protein structure including the M4 and M6 surfaces that interact with bound ions. Such an effect on ion binding would be consistent with the small, abnormal Na + transient currents seen with p.V130del.…”