Fig. 7: Identification of key and conserved TRPA1 DCTCaMBE residues involved in Ca²⁺/CaM binding.

a Homology model of TRPA1 distal C-terminus (residues 1100–1114, yellow) bound to Ca²⁺/CaM. WT CaM atomic model with N-lobe (dark green, residues 1–79) and C-lobe (light green, residues 80–149) denoted. Homology model built with the Modeller Comparative tool in ChimeraX with the TRPV1 distal C-terminal CaM binding segment (CT, blue)-Ca²⁺/CaM X-ray crystal structure as the template (PDB: 3SUI). Sequence alignment between TRPV1 and TRPA1 used for model building is in Supplementary Fig. 9a. This model yielded a z-DOPE score of 0.1256. Ca²⁺ ions bound to the four CaM EF hands are modeled in pink. b Ribbon diagram of homology model from (a). Key TRPV1 and TRPA1 residues involved in mediating these interactions are depicted as balls and sticks. c Immunoblotting analysis of the indicated 3xFLAG-hTRPA1 constructs after CaM-agarose pulldown in the absence or presence of Ca²⁺ from lysates of HEK293T cells transfected with 3xFLAG-WT, W1103A, L1107A, V1110A, W1103A/V1106A/L1107A/V1110A (Quad), or R1102A/K1111A (RK/AA) hTRPA1. Samples were probed as in Fig. 1c. d Quantification of CaM-agarose pulldowns represented in (c). Pulldown was normalized to the WT hTRPA1 with Ca²⁺ average. Data represent mean ± SEM. P values indicated in figure panel. n = 3 independent experiments, one-way ANOVA with Tukey’s post hoc analysis. e Sequence alignment of nine TRPA1 orthologues aligned to residues 1089–1112 of hTRPA1. Alignment generated with Sequence Logo. Box denotes TRPA1 DCTCaMBE. Hydrophobic (orange) and salt bridge (blue) residues proposed to form the DCTCaMBE are denoted with stars. f Immunoblotting analysis of the indicated MBP-tagged TRPA1 species orthologues after CaM-agarose pulldown in the absence or presence of Ca²⁺ from lysates of HEK293T cells transfected with MBP-WT mouse, zebrafish TRPA1 paralog a (zA1a), or C. elegans TRPA1 or the Δ1109–1119 equivalents of mouse (Δ1111–1125) or zebrafish (Δ1101–1115) TRPA1. Blot is representative of four independent experiments. Samples were probed using an anti-MBP primary antibody. Tubulin from whole cell lysates (10%, inputs) was the loading control.