# Computational design of constitutively active mutants of Dopamine D2 receptor inspired by ligand-independent activation mechanisms

## TL;DR

This paper uses computational methods to design and validate a new constitutively active mutant of the Dopamine D2 receptor, offering insights into receptor activation without ligands.

## Contribution

A novel computational-experimental framework for discovering constitutively active GPCR mutants, validated with a new D2R-I481.46W mutation.

## Key findings

- CAMs promote a conformational shift favoring an active state in D2R.
- A new single-point CAM, D2R-I481.46W, was identified and functionally validated.
- The mutation activates allosteric communication pathways involving transmembrane helix 5.

## Abstract

G protein-coupled receptors (GPCRs) can signal in the absence of agonists through constitutive activity. This activity can be enhanced by mutations, resulting in receptors known as constitutively active mutants (CAMs). Such receptors are implicated in various physiological and pathophysiological conditions, and also offer significant therapeutic potential. However, the molecular basis of their constitutive activity remains unknown.

To investigate how CAMs affect receptor activation, we employed enhanced sampling simulations to study the dopamine D2 receptor (D2R), a key target in central nervous system therapies. Free energy landscape analyses revealed that CAMs promote a conformational shift favoring an active state similar to the agonist-bound receptor. To then identify novel CAMs, we developed a comprehensive strategy combining structural comparison, in-silico residue scanning, and free energy calculations, validated by luminescence-complementation-based assays. Applied to D2R, this approach uncovered a new single-point CAM, D2R-I481.46W, which was functionally validated. Further investigation revealed that this mutation activates allosteric communication pathways primarily involving transmembrane helix 5, particularly Ser1945.43, underscoring its role in transmitting activation signals to the intracellular domain.

This study elucidates how CAMs reshape the activation landscape of D2R and establishes a broadly applicable computational-experimental framework for discovering constitutively active GPCR variants. These CAMs provide valuable ligand-independent models for probing receptor activation mechanisms at structural, cellular, and physiological levels.

The online version contains supplementary material available at 10.1186/s12915-026-02542-6.