G-protein coupled receptors (GPCRs)

Studying GPCR dynamics by NMR

G-protein coupled receptors play vital roles in health and disease, and hence represent a major class of drug targets. The β₁-adrenergic receptor binds endogenous catecholamine ligands such as adrenaline and regulates myocardial contraction via its class G_s heterotrimeric G-protein signalling partner.  Crystal structures of β₁AR and β₂AR bound to inverse agonists and full agonists show that ligand binding alone does not lend enough energy to lock the receptor into the active, G-protein binding conformation, characterised by a 14 Å outward movement of trans-membrane helix 6 as well as an extension of TM5.

This implies that β-adrenergic receptors are not simple on/off switches, but sample a complex energy landscape of activation. Such dynamics are well suited for investigation by NMR and transient intermediate states have been seen, which are not observed in lowest-energy-state crystal structures. Our research, using solution NMR, aims to study the importance of the GPCR conformational energy landscape to GPCR function and signalling including interaction of GPCRs with intracellular binding partners such as nanobodies and G_s mimics such as mini-G_s.

In order to study GPCRs by NMR, we use functional expression in insect cells, which yields sufficient quantities of material for structural biology research. Selective ¹³C labelling of methionine methyl groups provides reporters, distributed across the GPCR, which can be used to study the response of the GPCR under different conditions. An example of the reporters used in the β₁AR is shown below.

Selective mutagenesis is used to assign the NMR peaks arising from the different reporters, enabling the response of the receptor to different conditions e.g. different ligands, to be assessed.

(Above) [¹H,¹³C] spectra showing the response of methyl reporters in the β₁AR to different ligands and IBPs.

For more details see our recent publication on the β₁-adrenergic receptor:

Insight into partial agonism by observing multiple equilibria for ligand-bound and G_s-mimetic nanobody-bound β₁-adrenergic receptor.
Andras S. Solt, Mark J. Bostock, Binesh Shrestha, Prashant Kumar, Tony Warne, Christopher G. Tate, and Daniel Nietlispach, Nature Communications(2017)28, 1795
(doi: 10.1038/s41467-017-02008-y)