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).