ИСТИНА

TRPA1 channel belongs to the P-loop channels superfamily and contains a central pore domain and four voltage-sensor-like (S1-S4 or VSL) domains. In other P-loop channels membrane-embedded S1-S4 domains have short extracellular loops and spider toxins that affect their gating are often bind to these loops from the membrane side. However, the site of spider toxins binding to the TRPA1 channel is unknown. Pha1β (PnTx3-6) is a neurotoxin from the Phoneutria nigriventer spider, identified as a TRPA1 positive modulator. The toxin N-terminal domain contains an inhibitor cystine knot motif containing a long protruding loop, while C-terminal domain forms an extended α-helix. The total and selectively labeled variants of TRPA1 VSL domain were synthesized in the cell-free system. An almost complete 13C and 15N resonance assignment of the VSL backbone was obtained in the LPPG micelles environment. Titrations of the 15N-labeled VSL-TRPA1 with the unlabeled Pha1β and vice versa revealed the changes in the 1H and 15N chemical shifts of the domain and toxin, which evidenced the VSL/Pha1β complex formation in the LPPG environment. Largest changes were observed for the residues of S1–S2 and S2–S3 extracellular loops, S2, S3, and S4 helices of VSL domain and for the residues from the protruding loop and C-terminal α-helix of the toxin. A model of the Phα1β/TRPA1 complex was built using NMR data and a combined docking/molecular dynamics protocol. Phα1β binds to the S1-S2 loop of the VSL domain from the membrane bound state and contacts the outer mouth of the pore domain via the C-terminal α-helix. We propose that the Pha1β binding at the extracellular side of the TRPA1 channel to the interface of VSL and pore domains, changes the packing of these domains. It affects the properties of the lipid-binding site at the intracellular side of the channel, which modulates the opening and closing of the pore.