Figures and data
Cryo-EM structures of nhTMEM16 in the MSP1E3 and MSP2N2 nanodiscs.
a-b, Cryo-EM maps of nhTMEM16 in nanodiscs formed from a 7:3 mix of DOPC:DOPG lipids and MSP1E3 (a-c) or MSP2N2 (d-e) scaffold proteins. (a) Ca2+-free, closed groove, (b) Ca2+-bound, closed groove, (c) Ca2+-bound, open groove, (d) Ca2+-bound, intermediate-open groove, and (e) Ca2+-bound, open groove. In all dimers, one protomer is colored in gray and the other in blue (a), cyan (b), red (c), yellow (d), and sand (e). f-j, The groove in the different conformations is viewed from the plane of the membrane. Transmembrane helices are shown in cartoon representations and labelled. States are colored as in (a-e). Colored spheres correspond to the position of the Cα atoms of I334 on TM4 and Y439 on TM6 and their distance is indicated. Ca2+ ions are shown as green spheres. k, The percentage of particles with a closed (black bar), intermediate (gray bar), or open (white bar) groove conformation in the datasets of Ca2+-bound nhTMEM16 in MSP1E3 or MSP2N2 nanodiscs. Here, intermediate does not distinguish between the intermediate-open and -closed conformations. l-m, Alignment of the groove helices in the intermediate-open (yellow) and previously reported intermediate-closed (PBD: 6QMA, dark green) states (l), the distance between the Cα atoms of I334 on TM4 and Y439 on TM6 increases by ∼2 Å in the intermediate-open (m). n, The accessibility of the permeation pathway of nhTMEM16 in the intermediate conformation is visualized using the program Hole 39. o, The inner diameter of the permeation pathway of nhTMEM16 in MSP1E3 or MSP2N2 nanodiscs and in the published the diameter of a putative ion conduction pathway measured in different states with Hole including the Ca2+-bound open state in MSP2N2 (orange), Ca2+-bound open state in MSP1E3 (red), Ca2+-bound intermediate-open state in MSP2N2 (yellow), Ca2+-bound closed state in MSP1E3 (cyan), the previously reported intermediate-closed state in MSP2N2 (dark green) and Ca2+-free closed in MSP1E3 (blue).
Arrangement of lipids at the closed groove of nhTMEM16.
a-b, Segmented Cryo-EM map of nhTMEM16 in the Ca2+-bound closed state (gray), in MSP1E3 nanodiscs and DOPC/DOPG lipids, and the associated lipids (orange) viewed from the membrane plane (a) and from the extracellular side (b). The map showing the density of the nanodisc membrane is low-pass filtered to 10 Å and shown in transparent orange. c, View of the lipids outside of the closed groove from the plane of the membrane. d, Stick representation of the ten pathway lipids colored in yellow (P1-P10). Dashed arrow indicates the distance between the phosphate atoms of the last lipid from the inner (P6) and outer (P4) leaflets (∼27 Å) and the measured membrane thickness (∼39 Å). Lipids were built up to the phosphate atom in the head. Ca2+ ions are displayed as green spheres.
Identification of residues important for the closed groove scrambling.
a-c, Residues coordinating the P7-P9 (A), P3-P4 (B) and P2-P4 lipids (C) in the Ca2+-bound closed state. d-h, Representative traces of the dithionite induced fluorescence decay in the scrambling assay for protein free liposomes (green), or WT and the mutants in the presence (red) and absence (black) of Ca2+. h-i, Forward (α) and reverse (β) scrambling rate constants of the mutants of nhTMEM16 aimed at disrupting the protein-lipid interactions (shown in a-c) measured in 0 (h) or 0.5 mM Ca2+ (i). Bars are averages for α (black) and β (gray) (N ≥ 3), error bars are S. Dev., and red circles are values from individual repeats. The statistical significance of the effects of the mutants on the scrambling rate constants was evaluated with a two-sided Student’s t-test with a Bonferroni correction. **** denotes p<10-5 after Bonferroni correction.
Role of the E313-R432 salt bridge in groove opening.
a-b, Structural comparisons of the groove in apo (blue) vs Ca2+-bound closed (cyan) (a) and Ca2+-bound closed (cyan) vs open (red) states (b). Arrows denote rotations in the TM6. Sidechains of E313 and R432, and of the residues forming the TM4-TM6 interface are shown as sticks. The intra- and extra-cellular π-helical turns are colored in orange and gold, respectively. Ca2+ ions are displayed as green spheres. c, d, Cryo-EM maps of R423A nhTMEM16 in MSP1E3 (c) or MSP2N2 (e) nanodiscs. One protomer is colored in gray and the other in light blue (c) or magenta (e). The density of the nanodisc membrane is low pass filtered to 7 Å and shown in transparent orange (c) and red (e). d, f, Views of the groove from the plane of the membrane. TM4 and TM6 are shown in cartoon representation and labelled. Ca2+ ions are displayed as green spheres. g, Representative traces of the dithionite induced fluorescence decay in the scrambling assay for protein free liposomes (green), or R432A nhTMEM16 and the quadruple mutant R432A+Y327A/F330A/Y439A nhTMEM16 in the presence (dark blue and dark red) and absence (light blue and light red) of Ca2+. h-i, Forward (α) and reverse (β) scrambling rate constants of the mutants measured in 0.5 mM (h) or 0 Ca2+ (i). Bars are averages for α (black) and β (gray) (N ≥ 3), error bars are S. Dev., and red circles are values from individual repeats. The statistical significance of the effects of the mutants on the scrambling rate constants was evaluated with a two-sided Student’s t-test with a Bonferroni correction. ** denotes p<0.001 and **** denotes p< 10-5 after Bonferroni correction.
Disruption of TM6 straightening impairs lipid scrambling.
a, Representative traces of the dithionite induced fluorescence decay in the scrambling assay in protein-free liposomes (green) or proteoliposomes reconstituted with WT and A444P nhTMEM16 in the presence (dark blue and dark red) and absence (light blue and light red) of Ca2+. b-c, Forward (α) and reverse (β) scrambling rate constants of the mutants measured in 0.5 mM (b) or 0 Ca2+ (c). Bars are averages for α (black) and β (gray) (N ≥ 3), error bars are S. Dev., and red circles are values from individual repeats. The statistical significance of the effects of the mutants on the scrambling rate constants was evaluated with a two-sided Student’s t-test with a Bonferroni correction. **** denotes p<10-5 after Bonferroni correction. d-f, Cryo-EM maps of A444P nhTMEM16 in MSP1E3 nanodiscs in the long TM6 state (d), short TM6 state (e) and bent TM6 state (f). One protomer is colored in gray and the other in orange (d), pink (e), and purple (f). The density of the nanodisc membrane is low pass filtered to 7 Å and shown in transparent orange. g-i, The groove in the long TM6 (g), short TM6 (h) and bent TM6 (i) states is viewed from the plane of the membrane. Transmembrane helices are labelled and Ca2+ ions are displayed as green spheres.
The lipid environment determines the effect of mutations of nhTMEM16.
a-c, Representative traces of the dithionite induced fluorescence decay in the scrambling assay in protein-free liposomes (green) or proteoliposomes reconstituted with WT (a), R432A (b) or Y432A (c) nhTMEM16 in the presence (red) and absence (black) of Ca2+. Liposomes were formed from a 3:1 mix of DOPC/DOPG, or a 7:3 mix of POPE/POPG, DOPE/DOPG and POPC/POPG lipids. d-e, Forward (α) and reverse (β) scrambling rate constants of WT, R423A and Y439A nhTMEM16 in the four different lipid compositions (as in a-c) in the presence of 0.5 mM (d) or 0 Ca2+ (e). Bars are averages for α (black) and β (gray) (N ≥ 3), error bars are S. Dev., and red circles are values from individual repeats. The statistical significance of the effects of the mutants on the scrambling rate constants was evaluated with a two-sided Student’s t-test with a Bonferroni correction. *, **, and **** respectively denote p<5.10-3, <10-3, and <10-5 after Bonferroni correction.
Cryo-EM data collection, refinement and validation statistics
Structure determination of nhTMEM16 in the MSP1E3 nanodisc in 0 Ca2+.
a, Size exclusion profile of the reconstituted nhTMEM16-nanodisc sample in 0 Ca2+. The peak in the blue shadow contains the nhTMEM16-nanodisc complex. b, Representative micrograph. c, Representative 2D classes of the nhTMEM16-nanodisc complex. d, Angular distribution of the final reconstruction with C2 symmetry. e, Image processing workflow including symmetry expansion and 3D classifications of the signal subtracted monomers. Final masked reconstruction colored by local resolution calculated using the Relion implementation. f, FSC plots for nhTMEM16-nanodisc complex in 0 Ca2+. FSC (black) is between the two half maps to determine the resolution of the reconstruction evaluated at 0.143 cutoff. FSCsum (red), FSCwork (green), and FSCfree (blue) are model validations evaluated at 0.5 cutoff.
Structure determination of nhTMEM16 in the MSP2N2 nanodisc in the presence of Ca2+.
a, Size exclusion profile of the reconstituted nhTMEM16-nanodisc sample in the presence of 0.5mM Ca2+. The peak in the blue shadow contains the nhTMEM16-nanodisc complex. b, Representative micrograph. c, Representative 2D classes of the nhTMEM16-nanodisc complex. d, Angular distribution of the final reconstruction of the Ca2+-bound intermediate-open (top) and Ca2+-bound open (bottom) state. e, Image processing workflow. Final masked reconstruction colored by local resolution calculated using the Relion implementation. f, g, FSC plots for nhTMEM16-nanodisc complex in + Ca2+ in the MSP2N2 nanodisc. FSC (black) is between the two half maps to determine the resolution of the reconstruction evaluated at 0.143 cutoff. FSCsum (red), FSCwork (green), and FSCfree (blue) are model validations evaluated at 0.5 cutoff.
Structure determination of nhTMEM16 in the MSP1E3 nanodisc in the presence of Ca2+.
a, Size exclusion profile of the reconstituted nhTMEM16-nanodisc sample in the presence of 0.5mM Ca2+. The peak in the blue shadow contains the nhTMEM16-nanodisc complex. b, Representative micrograph. c, Representative 2D classes of the nhTMEM16-nanodisc complex. d, Angular distribution of the final reconstruction of the Ca2+-bound closed (top) and Ca2+-bound open (bottom) state. e, Image processing workflow including symmetry expansion and 3D classifications to identify monomers with well-resolved density for lipids to assist model building, and the open/closed dimers with one open and one closed groove. Final masked reconstruction colored by local resolution calculated using the Relion implementation. f, g, FSC plots for nhTMEM16-nanodisc complex in + Ca2+ in the MSP1E3 nanodisc. FSC (black) is between the two half maps to determine the resolution of the reconstruction evaluated at 0.143 cutoff. FSCsum (red), FSCwork (green), and FSCfree (blue) are model validations evaluated at 0.5 cutoff.
Classification of the symmetry-expanded protomers of WT nhTMEM16 in MSP1E3 nanodiscs from the particles of which the density around the groove region was not well resolved.
Structural characterization of the nhTMEM16 structures.
a-c, Structural alignment of nhTMEM16 structures determined in DOPC/DOPG lipids and MSP1E3 nanodiscs in apo (blue) and Ca2+-bound closed state (cyan) to the previously reported putative apo 6QM4 conformation (gray). nhTMEM16 is viewed from the plane of the membrane from the side. Horizontal arrows at the bottom denote the ∼4 Å displacement of the cytosolic domains away from the symmetry axis between the apo state and 6QM4. Vertical arrows denote the partial straightening of TM6 from the apo state to 6QM4. Superpositions of structures in panels a-c yield a Cα r.m.s.d of 1.23 Å (a), 0.67 Å (b), and 1.76 Å (c), respectively. d-e, Views of the Ca2+-binding site in the Ca2+-free state of nhTMEM16 from this study (d) (blue), the previously reported putative apo structure (PDBID: 6QM4, gray) (e) and the Ca2+-bound closed state from this study (f). The density maps (contoured at 4.5 σ) are shown in orange. Weak residual density at the center of the Ca2+-binding site is observed in 6QM4 (e). g, Structural alignment of the nhTMEM16 groove region of the structures in apo and Ca2+-bound closed state from this study and 6QM4. An intermediate state of TM6 is observed in 6QM4. h, i, Comparison of the groove in the open state obtained in DOPC/DOPG lipids in MSP1E3 (h) or MSP2N2 (i) to the previously reported open structure determined in POPC/POPG lipids and MSP2N2 (PDBID: 6QM9) (olive). j, Comparison of the groove in the intermediate-open state in MSP2N2 (yellow) to the previously reported intermediate-closed structure (PDBID: 6QMA) (dark green).
Lipid densities associated with nhTMEM16 in the Ca2+-bound closed state.
a, Structure model of nhTMEM16 in the Ca2+-bound closed state. Protomers in the dimer are colored in gray and cyan. Acyl chains of the built lipids are colored in yellow (at the closed groove) or magenta (at the dimer cavity) in one protomer and in gray in the other one. b-d, Close-up views of lipids at the closed groove (b) and the dimer cavity (c and d). Lipids are shown with mesh from the sharpened map in blue with σ=2.0. Respective lipids are labelled and Ca2+ ions are displayed as green spheres.
Structural comparison of nhTMEM16 apo with the Ca2+-bound closed state.
a, Alignment of the apo (blue) with the Ca2+-bound closed nhTMEM16 (cyan). b, Close-up view of the groove. Ca2+ ions are displayed as green spheres. d-e, Close-up views of the alignment of the residues coordinating lipids outside the groove in the Ca2+-bound state with the equivalent residues in the apo state. Side chains are shown as sticks. Representative transmembrane helices are labelled. f, g, Conformational arrangements on the TM6 when transits from apo state (blue) to the Ca2+-bound closed state (cyan). π-helical turn is colored in orange.
Structure determination and characterization of R432A nhTMEM16 in the MSP1E3 or MSP2N2 nanodisc in the presence of Ca2+.
a, Size exclusion profile of the reconstituted the R432A nhTMEM16-nanodisc sample in the presence of 0.5mM Ca2+. The peak in the blue shadow contains the R432A nhTMEM16-nanodisc complex. b, Representative micrograph. c, Representative 2D classes of the R432A nhTMEM16-nanodisc complex. d, Angular distribution of the final reconstruction in C2. e, Image processing workflow including symmetry expansion and 3D classifications to identify potential alternate conformations. Final masked reconstruction colored by local resolution calculated using the Relion implementation. f, g, FSC plots for R432A nhTMEM16-nanodisc complex in + Ca2+ in the MSP1E3 nanodisc (f) or MSP2N2 nanodisc (g). FSC (black) is between the two half maps to determine the resolution of the reconstruction evaluated at 0.143 cutoff. FSCsum (red), FSCwork (green), and FSCfree (blue) are model validations evaluated at 0.5 cutoff. h-j, Structural comparison of R432A nhTMEM16 in the MSP1E3 nanodisc (light blue) to WT nhTMEM16 in the Ca2+-bound closed state (cyan) from the side (h) or front view (i). Colored spheres correspond to the position of the Cα atoms of E313 on TM3 and R432 (or A432) on TM6 and their distance is indicated (j). Ca2+ ions are shown as green spheres. k, i, Views of the Ca2+-binding site in the R432A nhTMEM16 in + Ca2+ in the MSP1E3 nanodisc (k) or MSP2N2 nanodisc (i). m, n, Views of the upper groove region of nhTMEM16 in the Ca2+-bound closed state (m) and R432A nhTMEM16 in the Ca2+-bound closed state (n).
Structure determination of A444P nhTMEM16 in the MSP1E3 nanodisc in the presence of Ca2+.
a, Size exclusion profile of the reconstituted the A444P nhTMEM16-nanodisc sample in the presence of 0.5mM Ca2+. The peak in the blue shadow contains the A444P nhTMEM16-nanodisc complex. b, Representative micrograph. c, Representative 2D classes of the A444P nhTMEM16-nanodisc complex. d, Angular distribution of the final reconstruction in C2. e, Image processing workflow including symmetry expansion and classification to identify the four different conformations, the long TM6, short TM6, bent TM6 and long TM6/short TM6 A444P. Final masked reconstruction colored by local resolution calculated using the Relion implementation. f-i, FSC plots for A444P nhTMEM16-nanodisc complex in the MSP1E3 nanodisc in the Ca2+-bound closed state with long TM6 (f), short TM6 (g), bent TM6 (h) and long TM6/ short TM6 (i). FSC (black) is between the two half maps to determine the resolution of the reconstruction evaluated at 0.143 cutoff. FSCsum (red), FSCwork (green), and FSCfree (blue) are model validations evaluated at 0.5 cutoff. j-l, Views of the Ca2+-binding site of the nhTMEM16 mutant A444P in the long TM6 state (j), the short TM6 state (k) and the bent TM6 state (l) with map density colored in orange, pink and purple, respectively. Side chains of the Ca2+ coordinating residues are shown as sticks and the Ca2+ ions are displayed as green spheres.
Comparison of the membrane thickness at open and closed groove and the conserved lipid/detergent binding sites and the salt bridge in mammalian TMEM16s.
a, Two undecyl-maltoside molecules (shown in blue) were resolved in the cryo-EM structure (PDB: 6R7X, shown in gray) of TMEM16K in the Ca2+-bound closed state. b, The resolved lipids P4 and P8 associated with the closed groove of nhTMEM16. c, Alignment of the closed state of nhTMEM16 with TMEM16K. Ca2+ ions are displayed as green spheres. d, Lipids (shown in blue) outside of the open groove of afTMEM16 (PBD:7RXH, shown in gray). The distance between the phosphate atoms of the heads of P3 and P4 in the outer leaflet and inner leaflet is ∼20 Å. e, Lipids (shown in yellow) outside of the closed groove of nhTMEM16 (shown in cyan). The distance between the phosphate atoms of the heads of P4 and P6 in the outer leaflet and inner leaflet is ∼27 Å. f, Alignment of the rearrangements of the lipids outside of the open with the closed groove. The ∼39 Å membrane thickness is defined by the distance between the phosphate atoms of the heads of lipids away from the groove region and perpendicular to the membrane. Ca2+ ions are displayed as green spheres. g, i, The cryo-EM structure of TMEM16F (PDB:6QP6) in the Ca2+-bound closed state (g) and the predicted model of TMEM16E (i) by SWISS-MODEL (Waterhouse, Bertoni et al. 2018) based on TMEM16F (PDB:6QP6). The conserved salt bridge in the two structures is denoted by a dashed circle. h, j, Closed-up views of the salt bridge of R478-E604 in TMEM16F (h) and R484-E609 in TMEM16E (i).
