Abstract
Activation of Ca2+-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove and that Ca2+ dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements underlying groove opening and how lipids reorganize outside the closed groove remain unknown. Here we directly visualize how lipids associate at the closed groove of Ca2+-bound fungal nhTMEM16 in nanodiscs using cryo-EM. Functional experiments pinpoint lipid–protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryo-EM structure determination.
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Data availability
The data that support this study are available from the corresponding author upon request. All models and associated cryo-EM maps have been deposited into the Electron Microscopy Data Bank (EMDB) and the PDB. The depositions include final maps, unsharpened maps, local refined maps and associated FSC curves. The accession codes are listed here and in Tables 1–3. WT nhTMEM16 in MSP1E3 nanodiscs in the absence of Ca2+: EMD-41477 and PDB 8TPM; WT nhTMEM16 in MSP1E3 nanodiscs in the presence of Ca2+ (closed state): EMD-41453, EMD-41457 (consensus map), EMD-41458 (monomer map) and PDB 8TOI; WT nhTMEM16 in MSP1E3 nanodiscs in the presence of Ca2+ (open state): EMD-41455 and PDB 8TOL; WT nhTMEM16 in MSP2N2 nanodiscs in the presence of Ca2+ (intermediate-open state): EMD-41478 and PDB 8TPN; WT nhTMEM16 in MSP2N2 nanodiscs in the presence of Ca2+ (open state): EMD-41454 and PDB 8TOK; R432A nhTMEM16 in MSP1E3 nanodiscs in the presence of Ca2+ (closed state): EMD-41479 and PDB 8TPO; R432A nhTMEM16 in MSP2N2 nanodiscs in the presence of Ca2+ (closed state): EMD-41480 and PDB 8TPP; A444P nhTMEM16 in MSP1E3 nanodiscs in the presence of Ca2+: EMD-41481 and PDB 8TPQ for the closed state with long TM6, EMD-41482 and PDB 8TPR for the closed state with short TM6, EMD-41483 and PDB 8TPS for the closed state with bent TM6, EMD-41484 and PDB 8TPT for the closed state with asymmetric TM6 (long TM6 or short TM6). Source data are provided with this paper.
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Acknowledgements
The authors thank members of the Accardi laboratory, H. Weinstein and G. Khelashvili for helpful discussions and suggestions. The work was supported by National Institutes of Health grants R01GM106717 and R35GM152012 (to A.A.) and National Science Foundation Graduate Research Fellowship GRFP1746886 (to O.E.A.). Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR and the National Institutes of Health National Institute of General Medical Sciences (GM103310). Part of the work was performed at NYU Langone Health’s Cryo-Electron Microscopy Laboratory (RRID SCR_019202) with the help of B. Wang and W. Rice and at the Cryo-EM Core Facility at Weill Cornell Medical College with the help of C. Fluck. Initial screening was performed at NYU Langone Health’s Cryo-Electron Microscopy Laboratory (RRID SCR_019202) and the Cryo-EM Core Facility at Weill Cornell Medical College.
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Z.F. and A.A. designed the experiments; Z.F. and O.E.A. performed experiments; Z.F., O.E.A. and A.A. analyzed the data; and Z.F. and A.A. wrote the paper. All authors edited the manuscript.
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Nature Structural & Molecular Biology thanks Angela Ballesteros and Raimund Dutzler for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 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.
Extended Data Fig. 2 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.
Extended Data Fig. 3 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.
Extended Data Fig. 4
Classification of the symmetry-expanded protomers of Ca2+-bound WT nhTMEM16 in MSP1E3 nanodiscs from the particles of which the density around the groove region was not well resolved.
Extended Data Fig. 5 Lipid and nanodisc structural rearrangements in nhTMEM16.
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 (PDB 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 (PDB 6QM9) (olive). j, Comparison of the groove in the intermediate-open state in MSP2N2 (yellow) to the previously reported intermediate-closed structure (PDB 6QMA) (dark green).
Extended Data Fig. 6 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-e, Close-up views of lipids at the closed groove (b) and the dimer cavity (c-e). 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.
Extended Data Fig. 7 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. c-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.
Extended Data Fig. 8 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).
Extended Data Fig. 9 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.
Extended Data Fig. 10 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 (PBD7RXH, 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-MODEL65 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).
Supplementary information
Supplementary Information
Supplementary Table 1.
Supplementary Video 1
3D reconstruction of Ca2+-bound closed nhTMEM16 in nanodiscs associated with lipids.
Supplementary Video 2
Morph movie illustrating the conformational transitions of the groove of nhTMEM16 from Ca2+-free closed to Ca2+-bound open.
Source data
Source Data Fig. 1
Source data used in Fig. 1o.
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Source data used in Fig. 3d–i.
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Source data used in Fig. 4g–i.
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Source data used in Fig. 5a–c.
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Source data used in Fig. 6a–e.
Source Data Extended Data Fig. 1 and Table 1
Source data used in Extended Data Fig. 1f.
Source Data Extended Data Fig. 2 and Table 2
Source data used in Extended Data Fig. 2f,g.
Source Data Extended Data Fig. 3 and Table 3
Source data used in Extended Data Fig. 3f,g.
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Source data used in Extended Data Fig. 8f,g.
Source Data Extended Data Fig. 9 and Table 9
Source data used in Extended Data Fig. 9f–i.
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Feng, Z., Alvarenga, O.E. & Accardi, A. Structural basis of closed groove scrambling by a TMEM16 protein. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01284-9
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DOI: https://doi.org/10.1038/s41594-024-01284-9