Lipids and ions traverse the membrane by the same physical pathway in the nhTMEM16 scramblase
- University of Illinois at Urbana-Champaign, United States
- Emory University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Membrane deformation induced by nhTMEM16.
(A) nhTMEM16 is shown as a molecular surface (extracellular side up) colored by residue type (blue, basic; red, acidic; green, polar; white, nonpolar) in a phospholipid bilayer composed of POPC and …
Figure 1—figure supplement 1
nhTMEM16 surface hydrophobicity.
Molecular surface representation of nhTMEM16 crystal structure (4WIT), colored according to the hydrophobicity scale of Kyte and Doolittle (Cyan = hydrophilic (−4.5), Orange = hydrophobic (4.5)), …
Figure 2 with 1 supplement
Features of the ‘aqueduct’ on nhTMEM16 surface.
(A) Representative snapshots of the aqueduct during an MD simulation with Ca2+-activated nhTMEM16. Aqueduct-lining helices TM4 and TM6 are colored by residue type. Phospholipid head groups in the …
Figure 2—figure supplement 1
Penetration of lipids into the aqueduct in the Ca2+-activated simulation.
Number of water oxygen atoms (blue), lipid head group heavy atoms (orange), and lipid tail carbon atoms (black) within the aqueduct are plotted vs. simulation time. Within the first 100–150 ns, …
Figure 3 with 3 supplements
Kinetics of lipid permeation.
(A) Translocation of lipids along the aqueduct measured as the z-positions of phosphorus atoms versus time. Each 100-ns time point is highlighted as a dot. Complete flipping of a lipid from the …
Figure 3—figure supplement 1
Fraction of time each amino acid forms hydrophobic contact (<4 Å) with a fatty acid acyl chain whose head group is inside the aqueduct.
Analysis is combined for the two subunits. Residues with >10% time in contact are shown.
Figure 3—Video 1
Full scrambling of a POPC lipid from inner leaflet to outer leaflet of the membrane through the aqueduct under equilibrium condition.
One subunit of the nhTMEM16 dimer is shown colored N- to C- terminus blue to red. Ca2+ ions are purple spheres. The simulation is 950 ns in duration. View from the plane of the membrane. The …
Figure 3—Video 2
Full scrambling of a POPC lipid from inner leaflet to outer leaflet of the membrane through the aqueduct under equilibrium condition.
One subunit of the nhTMEM16 dimer is shown colored N- to C- terminus blue to red. Ca2+ ions are purple spheres. The simulation is 950 ns in duration. View looking into the aqueduct from the plane of …
Figure 4 with 1 supplement
Lipid binding sites and effects of mutations in nhTMEM16.
(A) Volumetric map of phosphate occupancy extracted from the Ca2+-activated simulation is shown as orange wireframe contoured at isovalue 0.15 overlaid on the protein structure. The three phosphate …
Figure 4—figure supplement 1
Amino acid residue interaction with lipid head groups in the aqueduct.
(A) Coordinating residues surrounding each binding site are labeled. The protein ribbon is colored based on the frequency of each residue forming electrostatic interaction with the head groups …
Figure 5
Phospholipid scrambling and ionic currents in nhTMEM16.
(A) Phospholipid scrambling and ionic currents are stimulated in HEK cells transfected with nhTMEM16 and co-transfected with a plasmid encoding EGFP. Transfected cells were identified by green …
Figure 6
Effects of amino acid mutations on TMEM16F (A,C) and TMEM16A (B,D).
(A and B) Images of cells during phospholipid scrambling. Single cells were patch clamped and intracellular Ca2+ controlled by 200 µM Ca2+ in the pipet. The first image in each row is EGFP …
Figure 7 with 6 supplements
Ion permeation occurs through the same structural pathway of lipid scrambling.
(A) Full permeation of one Na+ ion from the extracellular side to the intracellular side of the membrane at −150 mV (top panel) and −250 mV (bottom panel). Coordination number (left y-axis) and …
Figure 7—figure supplement 1
Translocation of the permeant Na+.
(A and B) Left panels: Translocation of the permeant Na+ (at −150 mV (A) or −250 mV (B)) through the aqueduct, measured as its z-position versus permeation time. The coordinating residues during ion …
Figure 7—figure supplement 2
Na+ permeation at −500 mV.
(A) Distribution of Na+ permeation duration for the 24 permeation events at −500 mV. One third of the events took place within five ns. (B). The proportion of coordination shows the contribution of …
Figure 7—figure supplement 3
At higher voltages (−1 V), both Na+ influx (38 occurrences) and Cl- efflux (eight occurrences) were observed during a 90 ns simulation.
https://doi.org/10.7554/eLife.28671.017
Figure 7—figure supplement 4
Dilation of the aqueduct.
(A) Average center of mass (COM) distance between TM4 and TM6 in the normal (blue) and dilated (green) states for each subunit during the 700 ns simulation at −500 mV. The COM distance for the …
Figure 7—figure supplement 5
Comparison of the aqueduct conformation with different transmembrane potentials.
The average COM distances between TM4 and TM6 at −150 mV and −250 mV are similar to that of the crystal structure and simulation at 0 mV; the value at −500 mV is slightly larger, due to the …
Figure 7—Video 1
Movie shows part of the simulation trajectory (~200 to 400 ns) at −500 mV, during which multiple ion permeations were captured through the aqueduct in subunit I.
Phospholipid phosphorus atoms are tan, Na+ ions are red. TM4 and TM6 are colored by residue type.
Figure 8 with 4 supplements
POPS is more coordinated by protein than POPC during flopping.
(A) Full permeation of POPS from the inner leaflet to the outer leaflet was captured in subunit I (left) and II (right) during the 1700 ns simulation (1000 ns equilibrium followed by 700 ns …
Figure 8—figure supplement 1
Scrambling events captured during the 1700 ns simulation.
(A) Multiple full scrambling for POPS and POPC from the inner leaflet to the outer leaflet were captured in both subunits. Translocation of the scrambled lipid through the aqueduct was measured as …
Figure 8—figure supplement 2
POPS binding inside the aqueduct.
Representative snapshots showing the phosphate and serine of the POPS head group are both coordinated by residues inside the aqueduct during the scrambling. POPS is coordinated by interactions …
Figure 8—figure supplement 3
Comparison of POPS and POPC head group coordination from residues E496, R505 and R548 during the 1700 ns simulation.
https://doi.org/10.7554/eLife.28671.024
Figure 8—figure supplement 4
Lipid P-N dipole orientations for head groups inside the aqueduct (bin = 2 Å along z axis) over the simulations with different transmembrane potentials.
The angle is defined by the P-N vector with respect to z axis. The phosphate groups precede the choline (POPC) or amino (POPS) groups in the lower half of the aqueduct (up to 4 Å) with obtuse angles …
Figure 9 with 3 supplements
Difference between Ca2+-free and Ca2+-liganded nhTMEM16.
(A) Ca2+-free and Ca2+-liganded nhTMEM16 were structurally aligned with UCSF Chimera and the RMSD of each residue calculated using least squares. TM domains are indicated at the top and RMSD values …
Figure 9—figure supplement 1
Comparison of head group distributions in the Ca2+-activated and Ca2+-free simulations.
Normalized probability histograms of the phosphate count in the 20 Å-thick core region of the aqueduct in subunit I (left panel) and II (right panel) were calculated for the last 500 ns of the …
Figure 9—figure supplement 2
Overlaid structures of Ca2+-free (tan) and Ca2+-activated (sky blue) nhTMEM16 as shown inFigure 9.
Ca2+ ions are magenta. Ca2+-coordinating residues are represented as sticks.
Figure 9—Video 1
Movie shows a morph between the representative conformations of the Ca2+-free and Ca2+-activated states.
Gating residues F330, T333, L336, V337, Y439, and T443 are shown in spacefilled with atoms colored by element (red, oxygen; grey, carbon; white, hydrogen). The morph was created in UCSF Chimera …
Figure 10 with 1 supplement
Aqueduct hydration.
(A) Average water density of the Ca2+-activated (left panel) and Ca2+-free (right panel) simulations is shown as a transparent cyan surface contoured at 0.15 of bulk water density, overlaid on …
Figure 10—figure supplement 1
Gating region of the aqueduct.
Representative snapshots showing the top view of the gating region of the aqueduct (z > 10Å is removed for clarity) during the Ca2+-activated (left panel) and Ca2+-free (right panel) simulations. …
Figure 11
A model for Ca2+-activated lipid scrambling.
Top panels: view from the membrane. Bottom panels: view from extracellular space. Left: Ca2+-activated. Right: Ca2+-free. Hydration is shown in blue and Ca2+ ions are shown as purple spheres. In the …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.28671.033
