Identification of TRPA1 channel residues critical for DNFB binding
To identify residues critical for DNFB binding to TRPA1, we carried out the molecular docking of DNFB onto the cryo-EM structure of human TRPA1 (PDB: 3J9P) using the Rosetta modeling. As shown in Fig. 5a-b, the docking reveals that DNFB is confined near the short helixes (H2, H4 and H5) in the coupling domain that is involved in electrophile irritant sensing (Suo et al., 2020). The ortho-nitro group of DNFB are both bound through hydrogen bonds to Cys621 at the end of short helix H2 and Tyr658 in the loop between β1.2 and H4 (Fig. 5a and b). In particular, in the binding model of DNFB, fluorine atoms unique in DNFB interact with Tyr684 through halogen bond, thus fixing the orientation of para-nitro pointing to H2, which causes the hydrogen bond between the para-nitro and Glu625, the π-Alkyl interaction with Ala688 and the π-Sulfur interaction with Cys621 to maintain conformation and position of the ligand (Fig. 5b). There are also some aromatic amino acids, such as Tyr681, interacting with DNFB through weak van der Waals forces and seems to be inessential for the binding of DNFB to TRPA1.
To confirm that Cys621 and Tyr658 in hTRPA1 are required for the DNFB binding, we introduced glycine and alanine mutations of C621G, Y658A and E681A. As shown in Fig. 5, application of 10 µM DNFB was unable to elicit detectable currents of TRPA1C621G (Fig. 5d and h) and TRPA1Y658A (Fig. 5f and h) mutants. In contrast, DNFB at 10 µM elicited robust E681A currents (Fig. 5g and h). Interestingly, the 10 µM DNFB cannot evoke the current of the TRPA1E625A, suggesting that Glu625 is essential for activation of TRPA1 by DNFB (Fig. 5e and h).