Molecular architecture and gating mechanisms of the Drosophila TRPA1 channel

The transient receptor potential channel subfamily A member 1 (TRPA1) ion channel is an evolutionary conserved polymodal sensor responding to noxious temperature or chemical stimuli. Notably, the thermosensitivity of TRPA1 varies among different species or even different isoforms in the same species. However, the underlying molecular basis of its thermo-gating remains largely unknown. Here, we determine the structures of a heat-sensitive isoform of TRPA1 in Drosophila melanogaster in two distinct conformations with cryo-samples prepared at 8 °C. Large conformational changes are observed in the ankyrin repeat domain (ARD) and the coiled-coil domain between the two states. Remarkably, all 17 ankyrin repeats are mapped in the newly resolved conformation, forming a propeller-like architecture. Two intersubunit interfaces are identified in the amino (N)-terminal domain, and play vital roles during both heat and chemical activation as shown by electrophysiological analysis. With cryo-samples prepared at 35 °C, only one conformation is resolved, suggesting possible state transitions during heat responses. These findings provide a basis for further understanding how the ARD regulates channel functions, and insights into the gating mechanism of TRPA1.

verified by Coomassie-blue-staining SDS-PAGE, suggests a good solution behaviour of the sample. b. A cryo-EM image of the dTRPA1-A isoform. Representative particles are indicated by green circles. c. Gold-standard Fourier shell correlation curves for the overall maps of two states. d. Euler angle distribution of the final 3D refinement of state-1. e, Local resolution map of state-1 calculated using ResMap.
f. FSC curves of the refined model versus the overall map of state-1 that it was refined against (magenta); of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (red); and of the model refined in the first of the two independent maps versus the second independent map (orange). g.
Euler angle distribution of the dTRPA1-A state-2 map. h. Local resolution map of the dTRPA1-A state-2 map. i. FSC model curves of the dTRPA1-A state-2 map. j.
Flowchart for cryo-EM data processing. Details can be found in the ''Image processing'' session in Methods. Inner panel, merge of the two EM-maps. k. Local resolution map of dTRPA1-A isoform captured at 35 C, as calculated using ResMap.
l. The gold-standard Fourier shell correlation curves for the overall map of dTRPA1-A isoform captured at 35 C. m. Structure alignments between the state-2 structure captured at 8 C (coloured marine) and the structure captured at 35 C (coloured red). a. Overall trend for the seventeen ARs observed in the dTRPA1 state-1 structure is quite different from that in dNOMPC (PDB ID: 5VKQ). One subunit of dTRPA1-A and dNOMPC is coloured orange and cyan, respectively. Two perpendicular reviews of the ARD alignment are shown on the right. b. Zoomed view of AR10 in the state-1 structure of dTRPA1-A isoform. AR10 is the turning point for the overall trend of the ARD in dTRPA1-A. The outer helix of AR10 is dramatically tilted compared to AR9 and AR11.

Supplementary Fig. S5 Structure comparison between dTRPA1-A and hTRPA1.
a. Structure alignments of the cytosolic domain alone between two states of dTRPA1-A. The nexus domain and the last five ARs aligns well between two states, suggesting that they may move in a rigid-body manner during state transitions. The state-1 and state-2 structures of dTRPA1-A are shown in grey and marine blue, respectively. b. Overview of the structure alignments between the two state structure of dTRPA1-A, the closed state of hTRPA1 (PDB ID: 6V9W, coloured green) and the activated state of hTRPA1 (PDB ID: 6V9X, coloured magenta). c. Structure alignments between dTRPA1-A and hTRPA1 for the VSLD. d. Structure alignments between dTRPA1-A and hTRPA1 for the pore region. e. Structure alignments between dTRPA1-A and hTRPA1 for the nexus region. f. Structure alignments between dTRPA1-A and hTRPA1 for the ARD. g. Structure alignments of the ARD alone reveal that the AR12-AR16 have an almost identical architecture. The N-terminus of AR12 is highlighted in a red circle. h. IFH (coloured magenta) and the succeeding loop insert into a pocket formed by the surrounding helices. i. Rotations of the pre-S1 and TRP helices between two states. j. Structure alignments of the IFH in dTRPA1-A and hTRPA1. Compared with the agonist JT010-bound hTRPA1 structure (PDB ID: 6PQO, coloured yellow) and the BITC-bound hTRPA1 structure (PDB ID: 6PQP, coloured pink), the IFH in the state-1 structure of dTRPA1-A (coloured grey) packs more tightly to nearby helices. The lipid molecule observed in the interfacial cavity of the JT010-bound hTRPA1 structure is shown in sticks. experiments were repeated six times (n = 6) for each construct. Significances were determined using a two-tailed unpaired t-test. **** P < 0.0001. Data are presented as mean ± s.e.m.

Supplementary Fig. S7 Expression of the WT dTRPA1-A isoform and the mutants in HEK293F cells.
Expression of WT dTRPA1-A isoform and all the mutants tested detected by western blot using anti-Flag tag antibody.

Supplementary Fig. S8 Reactive cysteine residues in dTRPA1-A.
a. Zoomed view of the structure alignment between two states of dTRPA1-A. State-1 is shown in grey and state-2 is shown in marine blue. Side chains of the key residues are shown in sticks. b. Sequence alignments between dTRPA1 isoforms and TRPA1 from human and rattlesnake. Shown here are the two segments containing the two reactive cysteine residues. c. Structure alignment of the nexus domain form hTRPA1 and dTRPA1-A alone. Side chains of the cysteine residues are shown in a ball-and-stick manner in the two state structure of dTRPA1-A, the closed state of hTRPA1 (PDB ID: 6V9W, coloured green) and the activated state of hTRPA1 (covalently modified by iodoacetamide, PDB ID: 6V9X, coloured magenta).
Structural shifts for the first helix-turn-helix motif are indicated by black arrows. d.
NMM-induced current densities at -60 mV for WT dTRPA1-A and the cysteine mutants. Independent experiments were repeated for at least four times for each construct (n = 6 for all, except C694K where n = 5). ns=not significant for WT versus mutants (one-way ANOVA with Dunnett's multiple comparisons test). Data are mean ± s.e.m. e. Heat-induced current densities for WT dTRPA1-A and the cysteine mutants.
Independent experiments were repeated for at least four times for each construct (n = 6 for all). ns=not significant for WT versus mutants (one-way ANOVA with Dunnett's multiple comparisons test). Data are mean ± s.e.m.

Supplementary Fig. S9 Model of the thermal activation of dTRPA1-A isoform.
a. Locations of the three residues in AR6 of dTRPA1-A isoform that are essential to thermal activation. b. Representative four-helix bundle structures in dTRPA1-A (state-2), TRPM4 (PDB ID:5WP6), TRPC6 (PDB ID:5YX9), KCNQ1 (PDB ID:5VMS) and the BacNaV channel NaVAb (PDB ID:5HK7). c. A cartoon model for the thermal activation of dTRPA1-A isoform. For thermal activation, TRPA1 is first sensitized from state-1 to a conformation like the state-2 structure of dTRPA1-A, and opens through movements of the TRP and pre-S1 helices in the nexus region. Supplementary Table S1