1. Structural Biology and Molecular Biophysics
  2. Computational and Systems Biology
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The selectivity of the Na+/K+-pump is controlled by binding site protonation and self-correcting occlusion

  1. Huan Rui
  2. Pablo Artigas
  3. Benoît Roux  Is a corresponding author
  1. The University of Chicago, United States
  2. Texas Tech University Health Sciences Center, United States
Research Article
Cite this article as: eLife 2016;5:e16616 doi: 10.7554/eLife.16616
7 figures and 6 tables

Figures

The ion binding sites in (A) Na3·E1·(ADP·Pi) (PDBID 3WGV) and (B) E2(K2) (PDBID 2ZXE) states.

Only the transmembrane helices M4, M5, M6, M8, and M9 from the α subunit are shown. Residues in the binding site are highlighted in stick presentation with those protonatable colored in yellow. Na+ (yellow) and K+ (magenta) ions are in spheres. The binding site number indices are presented on top of the ions. The view is from the extracellular side towards the intracellular side. The figure is produced with PyMOL (DeLano, 2002).

https://doi.org/10.7554/eLife.16616.003
Comparison of snapshots at the end of the MD simulations (green) and the crystal structure of Na3·E1·(ADP·Pi) (PDBID 3WGV) (white).

The binding site residues are shown in stick presentation and the ions are shown as spheres. Binding site Na+ ions from the MD simulation snapshots are in yellow, and the crystal Na+ are in orange. A Cl- ion has entered the binding site in system E1_S4 during the simulation and is shown in green. The view is from the extracellular side towards the intracellular side. The figure is produced with PyMOL (DeLano, 2002).

https://doi.org/10.7554/eLife.16616.007
Ion binding sites selectivity characterized by ln(KD,K/KD,Na) in states Na3E1·(ADP·Pi) (blue) and E2(K2) (red).

Sites I (square), II (circle), and III (triangle) are distinguished by their shapes. Values from the previous calculations with a smaller reduced region are shown as empty symbols. All the binding site glutamates (i.e., E327, E779, and E954) are kept protonated. The protonation states of the binding site aspartates are indicated below the plot.

https://doi.org/10.7554/eLife.16616.008
Charge-neutralizing mutations and their impact on binding site selectivity.

The wildtype protein is colored black and the mutations in E1 (blue) and E2 (red) are colored differently. Sites I (square), II (circle), and III (triangle) are distinguished by their shapes. The empty symbols represent values calculated from the outward facing P-E2 model.

https://doi.org/10.7554/eLife.16616.010
The binding site ion selectivity along the pump cycle.

Sites I (square), II (circle), and III (triangle) are distinguished by their shapes. Different colors indicate whether there are two (red) or three (blue) sites that are included in the calculations. The conformational states are numbered and stamped along the pump cycle in the top panel. The protonation states of the aspartates are indicated below.

https://doi.org/10.7554/eLife.16616.011
Water pathways from the cytoplasm to D926 in (A) Na2·E1·(ADP·Pi) and (B) P-E2·Na2.

The top (top) and side (bottom) views are shown. D926 are shown in sphere representation. Water path connecting the cytoplasm and the D926 are in surface representation colored in blue. Na+ (yellow) are shown as spheres.

https://doi.org/10.7554/eLife.16616.013
Fitting the potential of mean force 𝒲(χ) (red) with the boosting potential, UBP(χ) (black).
https://doi.org/10.7554/eLife.16616.015

Tables

Table 1

Atoms coordinating the binding site ions in the crystal structures and from the MD simulations. O is the backbone carbonyl oxygen atom. OG and OG1 are the hydroxyl oxygen atoms in serine and threonine. OD1 and OD2 are the carboxyl oxygen atoms in asparate. OE1 and OE2 are the carboxyl oxygen atoms in glutamate. OH2 is the water oxygen.

https://doi.org/10.7554/eLife.16616.004
E1Site ISite IISite III
x-rayMDx-rayMDx-rayMD
A323OT772OG1V322OE779OE1Y771OY771O
E779OE1T772OV325OD804OD1T774OT774O
D808OD1N776OD1E327OE2D808OD1Q923OE1Q923OE1
D808OD1D804OD1WaterOH2D926OD1
D808OD2WaterOH2
E2Site ISite II
x-rayMDx-rayMD
T772OS775OGV322OA323O
S775OGN776OD1V325OV325O
N776OD1D804OD2E779OE2E779OE1
D804OD2D804OD2D804OD1
Table 2

pKa values of binding site titratable residues calculated from the crystal structures. The crystal structure resolution is given below the PDB ID.

https://doi.org/10.7554/eLife.16616.005
E1E2
3WGV
2.8 Å
4HQJ
4.3 Å
2ZXE
2.4 Å
3B8E
3.5 Å
D8045.9(6.2)*11.1 (11.2)3.70.8 (2.1)
D8083.5(3.1)3.7 (3.7)5.86.8 (6.6)
D9266.4(7.2)5.6 (5.6)8.97.4 (8.4)
E32711.0(11.3)5.7 (5.6)8.310.8 (9.9)
E7799.9(8.4)7.4 (7.3)10.79.6 (8.0)
E9549.6(9.7)9.2 (9.2)10.310.7 (10.3)
  1. *If two chains are present in the same asymmetric unit, the pKa of the same residue in the other chain is shown inside the parenthesis.

Table 3

Summary of the all-atom simulation systems and the FEP/H-REMD reduced systems. The binding site residues E327, E779, and E954 were kept protonated in all the systems.

https://doi.org/10.7554/eLife.16616.006
SystemsBinding site residuesSimulation time (ns)
MDFEP/H-REMD
Wildtype
E1_S0D804-D808-D926-1402 × 128
E1_S1D804pD808-D926-3002 × 128
E1_S2D804-D808pD926-1392 × 128
E1_S3D804-D808-D926p5032 × 128
E1_S4D804pD808pD926-94
E1_S5D804pD808-D926p87
E1_S6D804-D808pD926p277
E1_S7D804pD808pD926p141
E2_S0D804-D808pD926-1932 × 128
E2_S1D804-D808pD926p3502 × 128
P-E2_S0D804-D808pD926p1002 × 128
Mutants
E1_S1MD804ND808-D926-402 × 128
E1_S2MD804-D808ND926-402 × 128
E1_S3MD804-D808-D926N402 × 128
E2_S1MD804ND808-D926p402 × 128
E2_S2MD804-D808ND926p402 × 128
E2_S3MD804-D808-D926N402 × 128
P-E2_S1MD804ND808-D926p402 × 128
P-E2_S2MD804-D808ND926p402 × 128
Table 4

The binding free energy difference (ΔΔGNaK) at all the binding sites calculated from FEP/H-REMD simulations. The energy values are in kcal/mol.

https://doi.org/10.7554/eLife.16616.009
SystemsBinding sites
IIIIII
Wildtype
E1_S00.1 ± 0.14.5 ± 0.11.8 ± 0.1
E1_S13.7 ± 0.21.7 ± 0.14.7 ± 0.1
E1_S2-3.5 ± 1.03.8 ± 0.32.9 ± 0.6
E1_S3-1.5 ± 0.24.7 ± 0.4-0.5 ± 0.3
E2_S0-4.4 ± 0.3-6.1 ± 0.0
E2_S1-3.5 ± 0.1-6.1 ± 0.1
P-E2_S0-1.0 ± 0.60.7 ± 0.4
Mutants
E1_S1M1.4 ± 0.15.7 ± 0.02.4 ± 0.1
E1_S2M4.3 ± 0.76.6 ± 0.65.2 ± 0.9
E1_S3M-0.6 ± 0.11.7 ± 0.10.0 ± 0.0
E2_S1M-3.3 ± 0.3-5.7 ± 0.0
E2_S2M-5.4 ± 0.1-6.4 ± 0.1
E2_S3M-3.8 ± 0.1-7.2 ± 0.1
P-E2_S1M0.8 ± 0.91.9 ± 0.4
P-E2_S2M1.6 ± 0.51.9 ± 0.4
Table 5

Selectivity in the form of at the binding sites along the pump cycle from state P-E2·K2 to P-E2·Na3. The energies are in kcal/mol.

https://doi.org/10.7554/eLife.16616.012
*p/--/pp/pp/pp/p
s/--/ss/pp/ss/s
(1) P-E2.K2-0.7 ± 1.00.1 ± 0.5-1.0 ± 0.60.7 ± 0.4-0.6 ± 0.7
(2) Intermediate0.0 ± 0.41.8 ± 1.3-0.5 ± 0.3-5.1 ± 0.3-4.0 ± 0.9
(3) E2(K2)-4.9 ± 0.8-3.0 ± 0.5-5.0 ± 0.2-4.2 ± 0.1-9.3 ± 0.5
(4) Intermediate0.6 ± 0.7-0.8 ± 0.32.0 ± 0.3-2.7 ± 0.2-1.6 ± 0.4
(5) K2.E11.5 ± 0.31.1 ± 0.21.1 ± 0.1-3.8 ± 0.3-1.2 ± 0.5
(6) K2.E1*0.6 ± 0.50.7 ± 0.41.3 ± 0.2-0.4 ± 0.80.1 ± 0.7
*s/-/--/s/-s/s/-s/s/-s/s/-s/s/ss/s/ss/s/ss/s/ss/s/ss/s/ss/s/s
p/-/--/p/-p/s/-s/p/-p/p/-p/s/ss/p/ss/s/pp/p/sp/s/ps/p/pp/p/p
(7) Na3·E1(ADP·Pi)-1.0 ± 0.51.7 ± 0.20.7 ± 0.72.2 ± 0.23.0 ± 1.00.8 ± 0.52.6 ± 0.36.0 ± 0.32.1 ± 1.55.8 ± 1.17.4 ± 0.87.5 ± 2.0
(8) Intermediate2.9 ± 1.0-0.5 ± 1.21.5 ± 1.62.6 ± 1.32.5 ± 1.33.0 ± 0.70.6 ± 0.20.8 ± 0.54.0 ± 0.82.8 ± 1.12.7 ± 0.53.5 ± 1.5
(9) P-E2·Na32.3 ± 0.31.6 ± 0.52.1 ± 0.32.4 ± 0.34.6 ± 0.51.4 ± 0.31.7 ± 0.3-2.2 ± 0.33.1 ± 0.8-0.6 ± 0.6-0.8 ± 0.6-1.2 ± 1.2
  1. *The top and bottom rows represent the starting and ending binding site ion configurations. A 'p' represents a K+ (potassium) ion and an 's' represents a Na+ (sodium) ion. The binding sites I, II, and III in this order are separated by '/'.

Table 6

Binding site residues and the fitted parameters ki and χ0,i.

https://doi.org/10.7554/eLife.16616.014
k1χ0,1k2χ0,2k3χ0,3k4χ0,4k5χ0,5k6χ0,6
M4
E327p-0.93253.111.05561.081.84462.05-0.31256.440.39289.040.22582.28
M5
Y771-2.1962.511.21989.272.4660.910.66240.850.72144.750.63950.42
T774-4.2535.822.437101.33.60261.711.12844.861.07191.960.85776.09
S7753.068-26.370.12438.671.29162.22
N7762.664-166.230.91887.151.70962.85-0.5371.79-0.49550.76-0.40675
E779p-2.50829.171.79288.012.59162.280.48724.810.47673.740.46253.74
M6
D804-3.895-172.950.48980.041.89760.56-0.19782.91-0.27182.85-0.19379.47
D804p2.968-157.980.53281.761.83560.45-0.24731.05-0.29689.84-0.27883.37
N8042.981-160.180.371841.45159.42
D808-3.215-159.940.2157.871.74459.13
D808p-2.29855.971.20466.882.4462.530.74637.740.71555.740.62763.72
N808-2.34429.851.77163.62.65464.990.91439.190.7358.370.6361.7
M8
Q923-2.94333.42.21787.952.67161.480.60374.20.53530.650.55144.23
D926-4.361-169.10.45495.511.90260.16-0.28392.02-0.30688.18-0.21585.37
D926p-2.70428.741.47793.382.74461.080.81658.990.85258.370.80958.93
N9263.674-157.180.47989.171.37761.78-0.62582.59

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