Structural basis of closed groove scrambling by a TMEM16 protein

Zhang Feng
Omar E. Alvarenga
Alessio Accardi author has email address

Department of Anesthesiology, Weill Cornell Medical College;
Physiology, Biophysics and Systems Biology Graduate Program, Weill Cornell Medical College
Department of Physiology and Biophysics, Weill Cornell Medical College;
Department of Biochemistry, Weill Cornell Medical College

https://doi.org/10.63204/9585.1

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Figures and data

Cryo-EM structures of nhTMEM16 in the MSP1E3 and MSP2N2 nanodiscs.
A-E, 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 ³⁶. 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) 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-E, 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 (D) or 0.5 mM 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. **** 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. π-helical turns are colored in orange. Ca²⁺ ions are displayed as green spheres. C, E, 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-B, Representative traces of the dithionite induced fluorescence decay in the scrambling assay in protein-free liposomes (green) or proteoliposomes reconstituted with WT (A) or R432A (B) 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. C-D, Forward (α) and reverse (β) scrambling rate constants of WT, R423A and Y439A nhTMEM16 in the four different lipid compositions (as in A-B) in the presence of 0.5 mM (C) or 0 Ca²⁺ (D). 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.