Identification of a drug binding pocket in TMEM16F calcium-activated ion channel and lipid scramblase

Figures

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  Fig. 1. Identification of three distinct states in drug-free TMEM16F. a Gaussian filtered density of the nanodisc and unsharpened density of the protein dimer. Unsharpened cryo-EM density and atomic model for three states with different conformations of TM6. Right, Ca2+ binding sites, with sharpened cryo-EM density in semitransparent outline and the residues depicted as sticks colored by heteroatom, in (b) monomer with extended TM6 and (c) monomer with kinked TM6. d Electrostatic surface of the asymmetric TMEM16F dimer, where white represents hydrophobic areas and blue and red correspond to positively and negatively charged regions, respectively.

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  Fig. 2. Cryo-EM analysis reveals asymmetry of the TMEM16F dimer. a Cryo-EM density of the asymmetric state of the TMEM16F dimer with the monomers colored blue and light blue, respectively, and the lipid densities in grey. The gaussian filtered cryo-EM density (semitransparent) reveals distortion of the lipid nanodisc. b Side view of a TMEM16F monomer (blue) highlighting the trail of lipids (grey) covering the TM region. c Front and side view of the atomic model of the asymmetric state of TMEM16F with Ca2+ atoms and glycans shown in green and red, respectively. The ion conduction channel identified by HOLE is represented by spheres colored in rainbow scale based on the local width of the channel, where red <1.5 Å and blue >7.5 Å.

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  Fig. 3. Niclosamide and 1PBC in the same hydrophobic groove of TMEM16F. Atomic model of the TM1-TM6 region of (a) Class 1 and (b) Class 2 of the drug-free TMEM16F, (c) niclosamide-bound TMEM16F and (d) 1PBC-bound TMEM16F. In each case, the additional cryo-EM densities found in the area are shown. Below, zoom into the TM1-TM6 groove with the residues shown as sticks and colored by heteroatom and the additional density found within the pocket shown in semitransparent outline. Structures of niclosamide and 1PBC as determined by computational docking using Glide are shown in purple and green, respectively. Bottom panels, differential effects of K370A and F374A mutations on TMEM16F-mediated (e) PS exposure (n = 84 for WT; 35 for F321A; 26 for K370A and 43 for F374A) and (f) Ca2+ influx (n = 162 for WT; 76 for F321A; 32 for K370A and 52 for F374A). At least three independent experiments have been performed for each condition, each with distinct cell populations to assess biological rather than technical variability. The mean ± SEM is shown along with the statistical significance determined by unpaired t-test (two-tailed) for each mutant as compared to the wildtype control (F321A: p = 0.0254;…

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  Fig. 4. Functional validation of the drug binding site in TMEM16F. Representative curves of live imaging of TMEM16F-dependent PS exposure (a–d); and Ca2+ influx (e–h). NC is negative control with stable cell line expressing mScarlet rather than TMEM16F tagged with mScarlet. Data are represented as mean ± SEM. Scattered dot plots of time of onset of TMEM16F-dependent PS exposure (i) n = 76 for wt Ctrl; 14 for wt Niclo 1 μM; 29 for wt Niclo 3 μM; 25 for wt Niclo 10 μM; 28 for wt 1PBC 1 μM; 25 for wt 1PBC 3 μM; 29 for wt 1PBC 10 μM; n = 23 for Triple Ctrl; 25 for Triple Niclo 1 μM; 22 for Triple Niclo 3μM; 30 for Triple Niclo 10 μM; 28 for Triple 1PBC 1 μM; 25 for Triple 1PBC 3 μM; 42 for Triple 1PBC 10 μM and Ca2+ influx (j) n = 31 for wt Ctrl; 15 for wt Niclo 1 μM; 12 for wt Niclo 3 μM; 9 for wt Niclo 10 μM; 22 for wt 1PBC 1 μM; 31 for wt 1PBC 3 μM; 15 for wt 1PBC 10 μM; n = 24 for Triple Ctrl; 11 for Triple Niclo 1 μM; 13 for Triple Niclo 3 μM; 6 for Triple Niclo 10 μM; 28 for Triple 1PBC 1 μM; 23 for Triple 1PBC 3 μM; 17 for Triple 1PBC 10 μM. Time of onset could not be determined for time courses with a linear rather than sigmoidal rise. At least three independent experiments have been…

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  Fig. 5. Functional validation of the drug binding site in TMEM16F. Representative curves of live imaging of TMEM16F-dependent PS exposure (a) and (c); and Ca2+ influx (e) and (g). Data are represented as mean ± SEM. Scattered dot plots of time of onset of TMEM16F-dependent PS exposure [(b) and (d)] n = 84 for wt Ctrl; 47 for wt Niclo; 58 for wt 1PBC; 35 for F321A Ctrl; 30 for F321A Niclo; 24 for F321A 1PBC; 26 for K370A Ctrl; 21 for K370A Niclo; 38 for K370A 1PBC; 43 for F374A Ctrl; 33 for F374A Niclo; 48 for F374A 1PBC; 39 for T606A Ctrl; 41 for T606A Niclo; 33 for T606A 1PBC; 69 for F685A Ctrl; 27 for F685A Niclo; 50 for F685A 1PBC, and Ca2+ influx [(f) n = 103 for wt Ctrl; 77 for wt Niclo; 63 for F321A Ctrl; 58 for F321A Niclo; 13 for K370A Ctrl; 7 for K370A Niclo; 35 for F374A Ctrl; 13 for F374A Niclo; 14 for T606A Ctrl; 13 for T606A Niclo; 15 for F685A Ctrl and 16 for F685A Niclo and (h) n = 59 for wt Ctrl; 48 for wt 1PBC; 13 for F321A Ctrl; 19 for F321A 1PBC; 19 for K370A Ctrl; 17 for K370A 1PBC; 17 for F374A Ctrl; 16 for F374A 1PBC; 13 for T606A Ctrl; 14 for T606A 1PBC; 16 for F685A Ctrl; 23 for F685A 1PBC] after chemical induction via 25 mM paraformaldehyde (PFA) and 2 mM…

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  Fig. 6. The drug binding pocket identified in TMEM16F. a Schematic representation of the TMEM16F dimer (light blue and blue) embedded in a lipid bilayer (grey), where Ca2+ atoms are shown as green circles and the inhibitors as a purple polygon and dotted black lines represent the closed ion conduction pore. b Structure of the drug binding pocket in TMEM16F with the side chains of the surrounding residues shown as sticks and the non-conserved residues highlighted in orange. Computationally docked structures of niclosamide and 1PBC are shown in purple and green, respectively. All atoms are colored by heteroatom.