# Structural and dynamic insights into the biased signaling mechanism of the human kappa opioid receptor

## TL;DR

This study explores how specific amino acids in the KOR affect signaling, aiming to develop better pain treatments with fewer side effects.

## Contribution

The study identifies key amino acids involved in β-arrestin recruitment and biased signaling in the KOR.

## Key findings

- CryoEM structures reveal the ligand binding mode in the activated KOR state.
- Four amino acids (K2275.40, C2866.47, H2916.52, Y3127.34) are crucial for β-arrestin recruitment.
- MD simulations show these mutants adopt conformations with reduced β-arrestin activity.

## Abstract

The κ-opioid receptor (KOR) is a member of the G protein-coupled receptor (GPCR) family, modulating cellular responses through transducers such as G proteins and β-arrestins. G-protein-biased KOR agonists aim to retain analgesic and antipruritic actions while limiting aversion and sedation. Aiming to inform G-biased KOR agonist design, we analyze signaling-relevant residues from structural and dynamic views. Here we show, using multiple complementary methods, shared residues that determine β-arrestin recruitment by nalfurafine and U-50,488H. Cryo-electron microscopy structures of the KOR-Gi signaling complexes identify the ligand binding mode in the activated state. Vibrational spectroscopy reveals ligand-induced conformational changes. Cell-based mutant experiments pinpoint four amino acids (K2275.40, C2866.47, H2916.52, and Y3127.34; Ballesteros–Weinstein numbering is shown in superscript) that play crucial roles in β-arrestin recruitment. Furthermore, MD simulations revealed that the four mutants tend to adopt conformations with reduced β-arrestin recruitment activity. Our research findings provide a foundation for enhancing KOR-mediated therapeutic effects while minimizing unwanted side effects by targeting specific residues within the KOR ligand-binding pocket, including K2275.40 and Y3127.34, which have previously been implicated in biased signaling.

Kappa-opioid receptors (KORs) influence pain and itch. Here, using cryoEM, spectroscopy, and cell tests, the authors show how specific amino acids shape KOR responses. These findings may guide development of pain treatments with fewer side effects.