Figure 4. The structure of TRPV3_{K169A 2-APB} exhibits changes in both transmembrane and cytoplasmic domains.

(A) One 2-APB molecule is bound to each protomer of the TRPV3_{K169A 2-APB} channel (magenta). 2-APB is found between the HLH_CD, Pre-S1_CD and TRP domains in a binding site defined by residues H417 in the HLH_CD, H426, H430, W433 in the Pre-S1_CD and W692, R693 and R696 in the TRP domain. All residues are shown in stick and red sphere representation. 2-APB is shown in stick and cyan sphere representation. (B) The S6 helix of TRPV3_K169A (blue) undergoes an α-to-π transition in the presence of 2-APB (magenta). (C) The α-to-π transition tightens the connection between S6 and the TRP domain. In the TRPV3_WT structure (orange), the TRP domain and the S6 are connected via a loop, but in the TRPV3_{K169A 2-APB} channel (magenta) the TRP domain and S6 form a continuous helical structure. In addition, the TRP domain exhibits a swivel in the TRPV3_{K169A 2-APB} structure. (D–E) The coil-to-helix transition in TRPV3_{K169A 2-APB} increases coupling between the cytoplasmic domains and the TRP domain. In the TRPV3_WT structure (orange) (D), the loop of AR5 (magenta surface) does not interact with the HLH_CD (orange surface). However, in TRPV3_{K169A 2-APB} (magenta) (E) the coil-to-helix transition in the distal CTD induces a conformational change in the loop of AR5 (magenta surface), coupling it to the HLH_CD (orange surface) and the TRP domain (light blue surface). 2-APB (cyan stick and surface representation) contributes to increased interactions between the TRP domain and Pre-S1_CD.

Figure 4—figure supplement 1. Cryo-EM data collection and processing, TRPV3_{K169A 2-APB}.

(A) A representative micrograph from TRPV3_{K169A 2-APB} data collection. (B) 3D reconstruction workflow. (C) Euler distribution plot. Red regions show the most represented views. (D) Local resolution estimate, calculated in Relion. (E) FSC curves calculated between the half maps (blue), atomic model and the full map (red) and between the model and each half-map (orange and green).

Figure 4—figure supplement 2. Electron density in the TRPV3_{K169A 2-APB} cryo-EM map.

Representative electron densities in the TRPV3_{K169A 2-APB} cryo-EM map. Densities are contoured at level 0.03 and radius 2. The inset in the S6 panel shows a close-up of the density around the π-helical turn contoured at level 0.02 and radius 2. The black solid lines represent the H-bonds in the α-helix, which are broken in the π-helical turn.

Figure 4—figure supplement 3. Comparison of the pore conformations of hTRPV3_WT, hTRPV3_{K169A 2-APB} and mTRPV3_Open.

Bottom-up view of the hTRPV3_WT (PDB ID 6MHO, orange), hTRPV3_{K169A 2-APB} (magenta) and mTRPV3_Open (PDB ID 6DVZ, green) pores. The conformation of hTRPV3_{K169A 2-APB} resembles that of the mTRPV3_Open (Overlay).

Figure 4—figure supplement 4. Coupling between the cytoplasmic and transmembrane domains.

(A) Top view of the coupling between the TRP domain (light blue), the CD (orange, Pre-S1_CD and HLH_CD) and the AR5 (magenta) in TRPV3_WT. (B) In the TRPV3_{K169A 2-APB} structure, the coil-to-helix transition in the CTD forces the loop AR5 to change conformation (magenta) and interact with HLH_CD (orange). This induces a swivel in the CD (orange) and the TRP domain (light blue). 2-APB (cyan spheres) further increases the coupling between the CD and the TRP domain. (C–E) Electron density around the CTD, AR5, CD and TRP domain in TRPV3_WT (C), TRPV3_K169A (D) and TRPV3_{K169A 2APB} (E) viewed from inside of the cytoplasmic vestibule. The density is contoured at level 0.02 in TRPV3_WT, 0.01 in TRPV3_K169A and 0.02 in TRPV3_{K169A 2-APB}.

Figure 4—figure supplement 5. The CTD in thermoTRPV structures.

(A) The cryo-EM map (EMD-8921) and atomic structure of the open mTRPV3 (PDB 6DVZ). Close-up view shows the fit of the CTD region into the electron density. (B) hTRPV3_{K169A 2-APB} fit into the cryo-EM map of the open mTRPV3 (EMD-8921). Close-up view shows that the electron density can more feasibly be built as a helix. (C) The cryo-EM map (EMD-9115) and atomic structure of the hTRPV3 apo, closed state (PDB ID 6MHO). (D) The X-ray crystallographic electron density and atomic structure of the apo closed rabbit TRPV2 (PDB ID 6BWM). (E) The cryo-EM map (EMD-5778) and atomic structure of the apo, closed rTRPV1 (PDB ID 3J5P). (F) hTRPV3_{K169A 2-APB} fit into the cryo-EM map of the open apo, closed rTRPV1 (EMD-5778).

Figure 4—figure supplement 6. APB binding in TRPV3_{K169A 2APB}.

(A) Non-assigned densities (red) in the VSLD cavity of TRPV3_K169A (green, left) and TRPV3_{K169A 2-APB} (purple, right). Electron densities are contoured at levels 0.015 and 0.03, respectively. (B) No electron density is present at the proposed third 2-APB binding site at the extracellular side of the VSLD (arrow). Electron density is contoured at level 0.03. (C) Binding of 2-APB (red stick representation) between the TRP domain (yellow) and Pre-S1_CD (green) in TRPV3_{K169A 2-APB} half-map 1 (magenta, contoured at 0.0175), half-map 2 (yellow, contoured at 0.0175) and full map (blue, contoured at 0.03).

Figure 4—figure supplement 7. APB and camphor response of proposed 2-APB binding site mutants.

(A) Graphical representation of the current response ratio of sub- (30 μM) and saturating 2-APB (300 μM) to camphor (10 mM) calculated as the mean from each biologically independent experiment. (WT: n = 5 biologically independent experiments; R487A: n = 4 biologically independent experiments; R487W: n = 4 biologically independent experiments; Y540W: n = 4 biologically independent experiments; E501G: n = 4 biologically independent experiments; Y540A: n = 4 biologically independent experiments; Y565A: n = 4 biologically independent experiments; H426A: n = 5 biologically independent experiments;). (B) Representative voltage ramp (−60 to +60 mV, 400 ms) traces to wash (gray), 30 (green) and 300 (red) μM 2-APB, and 10 mM camphor (blue). Only H426A possessed a lower 2-APB to camphor ratio compared to WT channels. (C) Protomer of mouse TRPV3 Y654A mutant bound to 2-APB (PDB ID 6DVZ) with three proposed binding sites highlighted (dotted boxes) (Singh et al., 2018). Mutated residues are shown in stick and sphere representation.