2019 Oct 31
Dynamic Modulation of the Lipid Translocation Groove Generates a Conductive Ion Channel in Ca
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Dynamic Modulation of the Lipid Translocation Groove Generates a Conductive Ion Channel in Ca
Both lipid and ion translocation by Ca
2+-regulated TMEM16 transmembrane proteins utilizes a membrane-exposed hydrophilic groove. Several conformations of the groove are observed in TMEM16 protein structures, but how these conformations form, and what functions they support, remains unknown. From analyses of atomistic molecular dynamics simulations of Ca 2+-bound nhTMEM16 we find that the mechanism of a conformational transition of the groove from membrane-exposed to occluded from the membrane involves the repositioning of transmembrane helix 4 (TM4) following its disengagement from a TM3/TM4 interaction interface. Residue L302 is a key element in the hydrophobic TM3/TM4 interaction patch that braces the open-groove conformation, which should be changed by an L302A mutation. The structure of the L302A mutant determined by cryogenic electron microscopy (cryo-EM) reveals a partially closed groove that could translocate ions, but not lipids. This is corroborated with functional assays showing severely impaired lipid scrambling, but robust channel activity by L302A.
Conflict of interest statement
The authors declare no competing interests.
Structural characteristics of states in the lipid translocation mechanism.
a Central panel: 2-D landscape representing all the trajectories of the WT nhTMEM16 (Supplementary Table 1) mapped with the tICA transformation in the space of the first two tICA eigenvectors (tIC WT 1 and tIC WT 2; see “Methods”). The lighter shades (green to yellow) indicate the most populated regions of the 2D space. The dynamics of the two subunits of the protein in each trajectory were analyzed separately. Microstates representing the most populated states in these simulations are indicated by the numbered circles and represent various stages in the lipid translocation process. Representative structures of microstates 1 and 3 are shown in the surrounding snapshots. In these models, TM4 and TM6 are labeled; the relevant groove residues appear in space-fill representations and are labeled (see also Supplementary Fig. 1A). The location of the initial conformation of the system (the 4WIS model) is indicated by the yellow circle marked Start. b Structural comparison of the position of groove helices TM3-TM6 (in cartoon) in microstates 1 and 3. Note the major repositioning of TMs 3 and 4 from microstate 1 (light gray) to microstate 3 (dark gray). c The probability distributions of the C α-C α distance between V337 and V447 residues in the ensemble of conformations from microstates 1 and 3. The vertical dashed lines represent the 8.5 Å distance cut-off used to define the occluded conformation of the groove. d The pore radius as a function of position along an axis perpendicular to the membrane (channel coordinate) for the X-ray structure of nhTMEM16 (PDBID 4WIS, red line), and for selected structures from microstate 3 (gray lines) representing 34 frames (separated from each other by at least 10 ns time interval) from the last 500 ns of a 15 μs MD simulation (WT6 in Supplementary Table 1). The EC and IC ends of the pore are indicated; Z = 0 Å corresponds to the location of the Cα atom of residue Q436 (red triangle symbol), the dot marks the position of the Cα atoms of residue Y439, and the diamond symbol that of V447. The calculations were performed with HOLE ( http://www.holeprogram.org/)
The time evolution of an MD trajectory shows that occlusion of the groove is triggered by lipid tail insertion.
a Projection (large colored dots) of the 15µs-long WT6 MD trajectory calculated for one of the nhTMEM6 subunits on the 2D tICA landscape from Fig. 1. The colors of the large dots indicate the time-frames in the evolution of the trajectory: darker colors (blue, cyan) are indicating the initial stages of the simulation, lighter colors dots (yellow, green) correspond to the middle part of the trajectory, and red shades show the last third of the trajectory. In this representative trajectory the system is seen to have evolved from microstates 1 to 4, and to 3. b The evolution of the C α-C α distance between residues V337 and V447 as a function of time in the WT6 trajectory. c Time evolution of the minimum distance between the lipid tail penetrating the groove and residues V337 (in red) and V447 (in blue). d Structures of representative snapshots showing the gradual occlusion of the groove in going from microstate 1 to 4 and to 3. The color code in ( d) is the same as in Fig. 1a; TM4 and TM6 are labeled. Note that the lipid tail present inside the groove in microstate 4 is absent in the ensemble of states in microstate 3, suggesting that once the occluding configuration was established, the lipid tail is no longer necessary for its relative stability
Hydration of the central region of the groove in the WT and mutant nhTMEM16.
a A snapshot of the nhTMEM16 (TMs 3–6) from the WT2 simulation showing the location of the central region of the groove (the tan shaded rectangle). The thick black lines surrounding the shaded rectangle indicate the z-axis positions of the C α atoms of residues T381 and Q436 (red spheres). b The frequency of finding a specific number of water molecules in the central region of the groove calculated from the full set of the WT nhTMEM16 (red), L302A (blue), and L302W (blue) trajectories (see “Methods” for the definition of water distribution in the groove and its different regions). For each construct, the data shown are averaged over the two protomers of the protein
TM3 and TM4 in nhTMEM16 are locked in hydrophobic interactions mediated by the L302/I343/L347 triad of residues.
a Two views, related by 90 ◦ rotation around the membrane normal, of the groove region in the WT nhTMEM16 protein, show the locations of L302, I343, and L347 (in space fill and labeled). Helices TM3-TM6 that line the groove are labeled, and colored red, blue, white and gray, respectively. b Histograms of minimal distance between residue pairs L302 and I343 (in red), and L302 and L347 (in black), in the simulation trajectories of WT nhTMEM16 (see also Supplementary Fig. 6)
Structural characteristics of occluded groove conformation in L302A nhTMEM16.
a Projection of all the L302A trajectories (purple symbols) onto the 2D tICA landscape of the wild-type system from Fig. 1a. The dynamics of the two subunits of the protein in each trajectory were considered separately in the analysis. The location of the initial conformation of the system (the 4WIS X-ray model) is indicated on the 2D tICA landscape by the yellow circle marked Start. From this configuration, the L302A system evolved towards the microstates denoted by a–c described in the other panels (see also Supplementary Fig. 9). b Structural representation of the initial conformation of the L302A system and of the representative conformations of microstates a, b, and c. In these structural models, the relevant groove residues appear in space fill representations, and are labeled. c The probability distributions of the C α-C α distance between V337-V447 residues in microstates a, b, and c. The vertical dashed lines represent the 8.5 Å distance cut-off used to define the occluded conformation of the groove. d Structural superpositions with respect to the X-ray structure of the nhTMEM16 (PDBID: 4WIS; in white), of the groove regions (helices TM3-TM6) from the occluded groove conformation in the WT nhTMEM16 simulations (in blue) and from the L302A simulations (in red). The simulated structures of the wild type and the L302A nhTMEM16 are represented by the centroids of the respective ensemble of conformations (i.e., for the wild type—microstate 3 in Fig. 1a; for the L302A—microstate a in Fig. 5a, b)
Functional consequences of mutations at the TM3/TM4 interface.
a, b Quantification of forward (α) and reverse (β) scrambling rate constants for WT and mutant nhTMEM16 in the presence ( a) and absence of Ca 2+ ( b) determined by fitting fluorescence traces to Eq. 1 (“Methods”). For the WT protein in the presence of Ca 2+ the rate constants could not be determined and were constrained to be 0.2 s −1 as previously described ,. c Quantification of the fraction of liposomes containing at least one active ion channel in the presence (red) and absence (black) of Ca 2+ using Eq. 3 (“Methods”). Error bars represent S.D. and the values of individual experimental replicates are indicated as symbols. n = 4–20 from 2 + independent preparations
Structure of nhTMEM16 L302A in lipid nanodiscs.
a Masked cryo-EM density maps of L302A nhTMEM16 in the presence of 0.5 mM Ca 2+. One monomer is shown in gray, the other is teal. b Atomic model of L302A nhTMEM16. One monomer is gray, in the other the cytosolic domain is orange, the permeation pathway is green, and the remainder of the protein is blue. Ca 2+ ions are shown as red spheres. c– f Structural comparison of the lipid pathway of wt (4WIS, left panels, gray) and L302A (right panels, teal) nhTMEM16. The color scheme is the same throughout the figure. c The lipid pathway (TM3-TM6) of the L302A mutant and WT (4WIS) viewed from the plane of the membrane. d– f Close up views of the structural rearrangements induced by the L302A mutation at the lipid pathway. The protein backbone is shown in ribbon representation and important residues are shown as sticks and colored in CPK yellow. d L302A disrupts the TM3/TM4 interface. Dashed lines indicate the distances between L302 on TM3 and I343/L347 on TM4. The distances respectively change from 3.7 to 7.4 Å and from 5 to 7.2 Å indicating that TM4 moves away from TM3. e Closure of the pathway to the membrane. The movement of TM4 towards TM6 enables the packing of the side chains of T333/V337 on TM4 and Y349/T443/V447 on TM6 to form a steric barrier to lipid entry. f Opening of the extracellular gate. The rearrangements induced by the L302A mutation cause the disengagement of E313 and E318 on TM3 from R432 on TM6. The R432-E313 distance increases from 4.9 Å (WT) to 8.9 Å (L302A) while the R432-E318 distance increases from 4.5 Å (WT) to 12.3 Å (L302A). g The diameter of the L302A groove was estimated using the HOLE program. Purple denotes areas of diameter d > 5.5 Å and green areas where 2.75 < d < 5.5 Å. h Top view of the lipid pathway. TM3–6 are shown as cartoon helices. Gray: Ca 2+-bound open nhTMEM16 (4WIS); pink: Ca 2+-bound closed nhTMEM16 (6QMB); yellow: Ca 2+-bound intermediate (6QMA); teal: L302A nhTMEM16
Structural similarity between the cryo-EM and computationally predicted models of the L302A nhTMEM16.
a Time-evolution of the backbone RMSD of the TM helices for one monomer of the L302A nhTMEM16 from a 2 µs-long MD simulation (Supplementary Table 1) with respect to either the cryo-EM model of L302A nhTMEM16 (red), or the X-ray structure of the WT nhTMEM16 from PDBID: 4WIS. b Superposition of the TM helices of L302A nhTMEM16 of the cryo-EM model (teal) with the average structure from the last 500 ns timeframe of the MD trajectory (gray). The backbone atom RMSD over all 10 TM helices is 1.2 Å. TM3, TM4, and TM6 helices are opaque and labeled, whereas the rest of the TM-s are shown in transparent. The Ca 2+ ions from the cryo-EM structure are shown as red sphere. c The pore radius as a function of position along an axis perpendicular to the membrane (channel coordinate) for the cryo-EM structure of L302A (red line), and for 50 evenly spaced frames from the last 500 ns of the 2 μs MD simulation of the L302A from ( a) (gray lines). The EC and IC ends of the pore are indicated, and Z = 0 Å corresponds to the location of the C α atom of residue Q436 (red triangle symbol), the dot marks the position of the C α atoms of residue of Y439, and the diamond symbol that of V447. The calculations were performed with the program HOLE ( http://www.holeprogram.org/). d Values of the minimal radius of the pore versus the value of the channel coordinate at which the pore radius is minimal. The calculations are from the HOLE profiles shown in ( c) (the red colored data point corresponds to the minimum in the cryo-EM structure profile; see red line in ( c))
Comparative analysis of the electrostatic characteristics of the groove.
a– c The electrostatic potential in the pore region of various TMEM16 constructs, obtained by solving linear Poisson-Boltzmann equation (see “Methods”) are depicted on the surface created by the [−1.0; 1.0] kcal/(mol e) range of values in the groove. The results are shown for: a TMEM16A (PDBID 5OYB); b the cryo-EM structure of L302A-nhTMEM16; and c wild-type nhTMEM16 (PDBID 4WIS). The electrostatic potential in the range of [−1.0; 1.0] kcal/(mol e)) is overlaid on the groove helices (TMs 3, 4, 6, and 7) of the respective structures. The Ca 2+ ions are shown as purple spheres. The locations of the EC and IC vestibules are marked. d The electrostatic potential along the pore axis from the calculations shown in ( a). The decreasing Z coordinate along the pore axis corresponds to EC → IC direction. The locations of selected relevant residues are marked with different symbols, with the colors specifying the TMEM16 construct as follows: Red = L302A-nhTMEM16; Blue = TMEM16A; and Green = wild-type nhTMEM16. With the respective colors, the symbols represent the following: Triangle = C α atom of Q436 (Q637 in TMEM16A); Dot = C α atom of Y439 (I641 in TMEM16A); and Square = Ca 2+. e The electrostatic profile along the pore axis in the cryo-EM structure of L302A-nhTMEM16 (red thick line), and in 50 evenly spaced frames from the last 500 ns of the 2 μs MD simulation of the L302A (gray lines, see also Fig. 8). As in ( b), the decreasing Z coordinate along the pore axis corresponds to EC → IC direction. The locations of selected relevant residues are marked with symbols following the same code as in ( b)
Structural and functional characteristics of the known conformations of the groove of Ca
2+-bound nhTMEM16 scramblase. The groove conformations shown are (from left to right): Ca 2+-bound closed (PDBID 6QMB); intermediate (PDBID 6QMA); ion-conductive (PDBID 6OY3, this study); membrane-exposed (PDBID 6QM9); and lipid-conductive (obtained from MD simulations). In all structures, TMs 3–6 are colored in red, blue, white, and silver, respectively, and are labeled
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The nhTMEM16 Scramblase Is Also a Nonselective Ion Channel
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A Pore Idea: The Ion Conduction Pathway of TMEM16/ANO Proteins Is Composed Partly of Lipid
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