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TRPV1 Structures in Nanodiscs Reveal Mechanisms of Ligand and Lipid Action

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TRPV1 Structures in Nanodiscs Reveal Mechanisms of Ligand and Lipid Action

Yuan Gao et al. Nature.

Abstract

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.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Reconstitution of TRPV1 into lipid nanodisc
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-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 2D 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, 2D 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).
Extended Data Figure 2
Extended Data Figure 2. Single-particle cryo-EM of unliganded TRPV1 in lipid nanodisc
a, Representative raw micrograph of apo TRPV1 in nanodisc. b, Fourier transform of image in (a). Note Thon rings are visible to up to 3Å. c, Gallery of 2D class averages. 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 3D 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 3D density map colored 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 the “free” curves indicate little effect of over-fitting.
Extended Data Figure 3
Extended Data Figure 3. Single-particle cryo-EM studies of agonist-bound TRPV1 in lipid nanodisc
a, Representative raw micrograph of TRPV1-RTX/DkTx in nanodisc. b, Fourier transform of image in (a). c, Gallery of 2D class averages. 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 3D 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 3D density map colored 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 the “free” curves indicate little effect of over-fitting.
Extended Data Figure 4
Extended Data Figure 4. Single-particle cryo-EM studies of antagonist-bound TRPV1 in lipid nanodisc
a, Representative raw micrograph of TRPV1/capsazepine complex in nanodisc. b, Fourier transform of image in (a). c, Gallery of 2D class averages. 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 3D 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 3D density map colored 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 the “free” curves indicate little effect of over-fitting.
Extended Data Figure 5
Extended Data Figure 5. Improved resolution for structures determined in nanodisc
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 significantly 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.
Extended Data Figure 6
Extended Data Figure 6. Newly resolved TRPV1 cytoplasmic region in nanodisc-stabilized structure
a, A region in 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).
Extended Data Figure 7
Extended Data Figure 7. Categories of lipid densities observed in TRPV1 structures
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).
Extended Data Figure 8
Extended Data Figure 8. Focused analysis of DkTx density map
a, Flow-chart showing procedures of focused 3D classification of DkTx and proximal regions (see Methods section for details). b, Atomic models for both knots of DkTx are superimposed on density maps (pink mesh).
Extended Data Figure 9
Extended Data Figure 9. Lipid co-factor and vanilloids at the vanilloid binding site of TRPV1
a, Chemical structure of phosphatidylinositol. b, Local environment of the phosphatidylinositol binding site may accommodate multiple phosphatidylinositide species with phosphate substituents at 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.
Figure 1
Figure 1. TRPV1 structures determined in lipid nanodisc
a, Side and top views of reference-free 2D class averages of TRPV1 in nanodiscs, showing transmembrane helices and lipid bilayer. b, Side and top views of 3D reconstruction of TRPV1-ligand-nanodisc complex. Individual channel subunits are color-coded with two molecules of DkTx (purple) atop the channel and a molecule of RTX (red) in the vanilloid binding pocket. Densities of the nanodisc (grey) and well-resolved lipids (blue) are also shown.
Figure 2
Figure 2. Structural details of tripartite toxin-channel-lipid complex
a, Sequence of DkTx (top) showing location of intramolecular disulfide bonds and finger-like loops formed primarily by residues conserved between toxin knots (orange). Hydrophobic residues enable fingers to penetrate the lipid bilayer by ~9Å (bottom). b, Schematic top down view showing antiparallel arrangement of two DkTx molecules (purple) binding at subunit interfaces of a TRPV1 homo-tetramer (subunits are color-coded). c, Cutaway view depicting one DkTx molecule interacting with two adjacent TRPV1 subunits (grey) and associated lipids (blue spheres; red and orange spheres depict phosphate head groups). Superimposed ribbon diagram (light blue) denotes location of transmembrane α-helices for one channel subunit. d, Detailed view of boxed region in (c) showing interactions between lipids and amino acid side chains from channel and toxin (dotted line, hydrogen bond). Helices from three neighboring channel subunits are color-coded as in (b).
Figure 3
Figure 3. Movement of protein and lipids associated with toxin binding
a, Movement of pore loop, pore helix, and part of S6 domain from closed (blue) to open (orange) states upon DkTx (purple) binding. Without such movement, one finger of DkTx would clash (yellow region) with the unliganded channel at the top of S6. Top down view (right) shows two DkTx molecules atop TRPV1 (grey density). Toxin binding is associated with lateral shifts of the pore helix and loop (arrows), as well as large rearrangements of aromatic side chains within these regions. b, Two annular lipids (shown in blue, with phosphate in orange and oxygen in red) at the channel-toxin interface undergo both lateral and vertical movements upon DkTx binding. Dashed lines mark original position of phosphate groups in the absence of toxin (left); arrows indicate displacement of lipids in the presence of toxin (right).
Figure 4
Figure 4. Shared binding pocket for phosphatidylinositol lipids and vanilloid ligands
a, Surface representation of TRPV1 (grey) in cutaway view revealing location of bound co-factor (blue). Superimposed ribbon diagram (yellow) denotes location of transmembrane α-helices for one channel subunit. Detailed view of boxed region shows how co-factor density (blue mesh) accommodates a molecule of phosphatidylinositol (PI). Positive and negative side chains from S4 and S4–S5 linker, respectively, can form ionic interactions with negatively charged phosphate or hydroxyl moieties on inositol ring. Helices from a neighboring subunit (light blue) are also shown. b, Density for resiniferatoxin (RTX, red mesh) is well fit by its atomic structure. Residues essential for RTX sensitivity (Y511, M547, T550) lie in close proximity to the ligand and can engage in electrostatic or hydrophobic interactions. Densities for PI and RTX define overlapping, but non-identical sites (also see Extended Data Fig. 9).
Figure 5
Figure 5. Structural rearrangements associated with vanilloid binding
a, Ribbon diagrams depicting relative locations of S4, S4–S5 linker, S6 and TRP domain helices in the presence of phosphatidylinositol (blue, left), resiniferatoxin (orange, middle), or capsazepine (gold, right). Vanillyl ring of RTX uniquely stabilizes interaction between Arg557 and Glu570 to facilitate movement of the S4–S5 linker away from the central axis of the channel (indicated by red arrows), thereby facilitating opening of the lower gate through coupled movements (indicated by black arrows).
Figure 6
Figure 6. Mechanistic models for TRPV1 activation
a, Proposed mechanism for DkTx action. Two hydrophobic fingers (purple and pink) of each ICK knot (joined by three intramolecular disulfide bonds, yellow lines) enable the toxin to partition into the lipid bilayer (grey shade) and subsequently target TRPV1. In the closed state, the upper pore region of the channel (orange, pore helix; thick line, pore loop) undergoes brief spontaneous excursions to an open state, enabling DkTx to dock. Several annular lipids (blue ellipse with zigzag tails) bind at the channel-toxin interface to further stabilize the open state through formation of a tripartite complex. Resident phosphatidylinositides (blue hexagon attached to red sphere with zigzag tails) in the vanilloid pocket may leave upon toxin binding to facilitate allosteric opening of the lower gate. b, Proposed mechanism for vanilloid agonist action. Phosphatidylinositide co-factor binds in vanilloid pocket to stabilize the channel in its closed state. Vanilloid agonist (red hexagon attached to grey ellipse) displaces phosphatidylinositide to facilitate formation of a salt bridge between Arg557 (dark blue branch) and Glu570 (red branch), consequently pulling the S4–S5 linker away from the channel’s central axis to open the lower gate. c, Heat may open the channel through a similar mechanism involving thermal displacement of resident phosphatidylinositides.

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