An NMR approach to study functional GPCRs obtained by directed evolution

• Applicant: Professor Dr. Franz Hagn
• Subject Area: Structural Biology ; Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 466160427

G-protein coupled receptors (GPCRs) are essential components of the signal transduction pathways between cells and thus are targets of around 60% of all currently marketed drugs. Despite a growing number of crystal and EM structures, the mechanism of GPCR-mediated signaling is still not sufficiently well understood as activation and switching properties of GPCRs cannot be fully explained by static snapshots. In this project, we will characterize the structural and dynamical changes in a GPCR upon binding to an antagonist, agonist or G-protein using solution-state NMR spectroscopy as a main tool. In order to be able to study such a receptor system by NMR at high resolution and quality, high-level stable isotope labeling schemes (esp. high deuteration and selective methyl labeling) are essential. Bacterial expression systems (such as E. coli) are best suited for these special requirements. However, until recently, the production yields of GPCRs in E. coli have been very low. This unfavorable situation was fundamentally changed by the application of directed evolution methods to GPCRs, rendering high resolution NMR studies on this essential membrane protein class feasible. In the proposed study, we will use the well-characterized neurotensin receptor subtype-1 (NTR1) as a model system to develop a strategy to investigate a stabilized GPCRs by solution-state NMR. Furthermore, we will apply a rational NMR-based approach to rescue the biased functionality of this mutated GPCR. Preliminary results provide a promising picture that relevant solution state NMR investigations can be conducted on the mechanism of NTR1 activation by small molecule ligands as well as a G-protein using a functional and stabilized receptor variant. These experiments will contribute to a better understanding of the allosteric switching properties of a GPCR as well as provide new approaches for the structural investigation of GPCRs by solution state NMR.

• DFG Programme: Research Grants