# Binding and Activating of Analgesic Crotalphine with Human TRPA1

## TL;DR

This study reveals how the analgesic peptide crotalphine activates the TRPA1 ion channel, offering insights into pain signal transmission and potential analgesic drug development.

## Contribution

The novel contribution is identifying Cys621 as the primary binding site for crotalphine in TRPA1 and elucidating its allosteric activation mechanism.

## Key findings

- Cys621 in TRPA1 is the primary binding site for crotalphine.
- Crotalphine binding induces structural changes in TRPA1's transmembrane domain.
- Allosteric modulation by crotalphine facilitates ion permeation through TRPA1.

## Abstract

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation.