When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids have both structural and regulatory roles. Here we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the rat TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting the release of bioactive lipids from a critical allosteric regulatory site.
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Electron Microscopy Data Bank
Protein Data Bank
The three-dimensional cryo-EM density maps of the TRPV1–nanodisc complexes without low-pass filter and amplitude modification have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-8118 (TRPV1–nanodisc), EMD-8117 (TRPV1–RTX/DkTx–nanodisc), EMD-8119 (TRPV1–capsazepine–nanodisc) and EMD-8120 (TRPV1–capsazepine in amphipol). Particle image stacks after motion correction related to TRPV1–nanodisc and TRPV1–RTX/DkTx–nanodisc have been deposited in the Electron Microscopy Pilot Image Archive (http://www.ebi.ac.uk/pdbe/emdb/empiar/) under accession number EMPIAR-10059. Atomic coordinates for the atomic model of TRPV1 in nanodisc, TRPV1–RTX/DkTx in nanodisc and TRPV1–capsazepine in nanodisc have been deposited in the Protein Data Bank under accession numbers 5IRZ, 5IRX and 5IS0.
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We thank our laboratory colleagues, past and present, for many helpful discussions and manuscript critiques, C. Paulsen and E. Green for helping with initial screening for nanodisc reconstitution, and E. Palovcak for providing scripts for focused classification. This work was supported by grants from the National Institutes of Health (R01NS047723, R37NS065071 and R01NS055299 to D.J., S10OD020054, R01GM098672, P01GM111126 and P50GM082250 to Y.C.). Y.C. is an Investigator with the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Extended data figures and tables
a, Size-exclusion chromatography of TRPV1 channel reconstituted into lipid nanodisc using MSP2N2. Void volume and peaks corresponding to TRPV1 and cleaved MBP are indicated. b, SDS–polyacrylamide gel electrophoresis (SDS–PAGE) of detergent-solubilized MBP–TRPV1 fusion protein and material from nanodisc reconstituted with TRPV1 following MBP cleavage (middle peak in a). Note the presence of both bands for TRPV1 and MSP2N2. c, Representative micrograph of negative-stained TRPV1–nanodisc sample showing mono-dispersed and homogeneous particles. d, Reference-free two-dimensional class averages of particles in c, revealing band-like density contributed by the lipid disc (side view) and tetrameric arrangement of channel subunits (top view). e, Two-dimensional class averages of the same protein reconstituted into MSP1E3 nanodisc, which is smaller in diameter. Note the extra space within the disc offered by MSP2N2 scaffold protein in d. The size of the class average window is 258 Å.
a, Representative raw micrograph of apo TRPV1 in nanodisc. b, Fourier transform of image in a. Note that Thon rings are visible at up to 3 Å. c, Gallery of two-dimensional class averages, with size of window as 233 Å. d, Slices through the unsharpened density map at different levels along the channel symmetry axis (numbers start from extracellular side). e, Euler angle distribution of all particles included in the calculation of the final three-dimensional reconstruction. Position of each sphere (grey) relative to the density map (green) represents its angle assignment and the radius of the sphere is proportional to the amount of particles in this specific orientation. f, Final three-dimensional density map coloured with local resolution in side and top views. g, Fourier shell coefficient (FSC) curves between two independently refined half maps before (blue) and after (red) the post-processing in RELION. h, FSC curves for cross-validation: model versus summed map (purple), model versus half map 1 (used in test refinement, cyan), model versus half map 2 (not used in test refinement, orange). Small differences between the ‘work’ and ‘free’ curves indicate little effect of over-fitting.
a, Representative raw micrograph of TRPV1–RTX/DkTx in nanodisc. b, Fourier transform of image in a. c, Gallery of two-dimensional class averages, with size of window as 233 Å. d, Slices through the unsharpened density map at different levels along the channel symmetry axis (numbers start from extracellular side). e, Euler angle distribution of all particles included in the calculation of the final three-dimensional reconstruction. Position of each sphere (grey) relative to the density map (green) represents its angle assignment and the radius of the sphere is proportional to the amount of particles in this specific orientation. f, Final three-dimensional density map coloured with local resolution in side and top views. g, FSC curves between two independently refined half maps before (blue) and after (red) the post-processing in RELION. h, FSC curves for cross-validation: model versus summed map (purple), model versus half map 1 (used in test refinement, cyan), model versus half map 2 (not used in test refinement, orange). Small differences between the ‘work’ and ‘free’ curves indicate little effect of over-fitting.
a, Representative raw micrograph of TRPV1–capsazepine complex in nanodisc. b, Fourier transform of image in a. c, Gallery of two-dimensional class averages, with size of window as 233 Å. d, Slices through the unsharpened density map at different levels along the channel symmetry axis (numbers start from extracellular side). e, Euler angle distribution of all particles included in the calculation of the final three-dimensional reconstruction. Position of each sphere (grey) relative to the density map (green) represents its angle assignment and the radius of the sphere is proportional to the amount of particles in this specific orientation. f, Final three-dimensional density map coloured with local resolution in side and top views. g, FSC curves between two independently refined half maps before (blue) and after (red) the post-processing in RELION. h, FSC curves for cross-validation: model versus summed map (purple), model versus half map 1 (used in test refinement, cyan), model versus half map 2 (not used in test refinement, orange). Small differences between the ‘work’ and ‘free’ curves indicate little effect of over-fitting.
Comparison of density maps (blue mesh) determined from nanodisc- and amphipol-stabilized TRPV1 at various regions of the channel facing the lipid bilayer or at the bilayer surface. Refined atomic models (gold, nanodisc; grey, amphipol) are fit to corresponding densities. Side-chain densities were considerably improved in nanodisc-stabilized TRPV1–DkTx/RTX structure (a, b), and notable improvement was also seen for unliganded (c, d) and capsazepine-bound (e, f) channels in nanodisc.
a, A region in the TRPV1 C terminus, previously unresolved in amphipol-stabilized structures (blue) is clearly resolved in the nanodisc-stabilized structure. b, Enlarged view of boxed region in a showing density map (blue mesh) and superimposed model (gold). Previously resolved TRP domain and N-terminal β-strands are depicted in ribbon diagram format (cyan).
a, Two continuous layers of density (blue) contributed by lipid head groups of bilayer within nanodisc are shown for apo channel (left) and channel in complex with RTX–DkTx (right). b, Atomic model of annular lipids could be built into well-resolved densities (blue mesh) surrounding the channel protein. DkTx is shown as ribbon diagram (pink). Top-down views show distribution of resolved annular lipids (blue) in inter-subunit crevices at the outer leaflet of the membrane. c, Well-resolved densities (blue mesh) in the structures representing a phosphatidylcholine molecule (left) and a phosphatidylinositol molecule (right). Transmembrane helices of TRPV1 close to the binding site are also shown as ribbon diagrams (grey).
a, Flow-chart showing procedures of focused three-dimensional classification of DkTx and proximal regions (see Methods for details). b, Atomic models for both knots of DkTx are superimposed on density maps (pink mesh).
a, Chemical structure of phosphatidylinositol. b, Local environment of the phosphatidylinositol-binding site may accommodate multiple phosphatidylinositide species with phosphate substituents at the 3, 4 and/or 5 positions of the inositol ring (drawn in red). Adjacent regions of the channel are shown as ribbon diagram (grey). c, Tyr511 assumes two possible orientations that differ in apo versus agonist-bound states of the TRPV1 channel. In the apo state, one acyl chain of the resident phosphatidylinositol lipid (blue mesh superimposed with atomic model) prevents the Tyr511 side chain from assuming the upward rotamer position. d, Density maps of vanilloids (resiniferatoxin, red mesh; capsazepine, gold mesh) superimposed with density of the bound phosphatidylinositol lipid (blue mesh), suggesting that they occupy overlapping, but not identical sites. Atomic models for both drugs and their chemical structures are also shown. e, Overlap of transmembrane region of one TRPV1 subunit corresponding to apo (blue) and RTX/DkTx-bound (orange) states. Note the relatively small conformational change of the voltage sensor-like domain (S1–S4, boxed region). f, Overlap of transmembrane region of one TRPV1 subunit corresponding to apo (blue) and capsazepine-bound (gold) states.
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Gao, Y., Cao, E., Julius, D. et al. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534, 347–351 (2016). https://doi.org/10.1038/nature17964
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