Structure-guided optimization of light-activated chimeric G-protein-coupled receptors

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• G-protein-coupled receptors (GPCRs) are a vast human receptor family crucial for physiological processes.
• GPCRs exhibit specific coupling to signaling pathways, a feature that can be tuned for novel receptor functions.
• Recent high-resolution structures of GPCR-G-protein complexes offer opportunities for rational receptor engineering.

Purpose of the Study:

• To demonstrate structure-guided optimization of light-activated OptoXRs.
• To investigate the hypothesis that incorporating GPCR-Gα contacts improves receptor-G-protein coupling.
• To develop a platform for engineering OptoXRs with enhanced signaling and spectral tuning for targeted manipulation.

Main Methods:

• Evaluated structure-based alignments for chimeric receptor fusion.
• Engineered a light-activated beta-2 adrenergic receptor (β₂AR) by incorporating GPCR-Gα contacts.
• Assessed signaling function of engineered OptoXRs and performed spectral tuning.

Main Results:

• Incorporating Gα contacts into a light-activated β₂AR increased signaling 7- to 20-fold compared to other designs.
• Elimination of these contacts diminished receptor function, validating their importance.
• The developed platform enabled optimization of another OptoXR and allowed for spectral tuning.

Conclusions:

• Structure-guided engineering by incorporating GPCR-Gα contacts significantly enhances OptoXR signaling.
• This approach provides a powerful platform for developing novel OptoXRs with tailored functions.
• Optimized OptoXRs can be utilized for precise cell and network manipulation.