Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state

Abstract

Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.

Allosteric ligands control the activation of a class C GPCR heterodimer by acting at the transmembrane interface

“…First, GB1 TMD plays a key role for the agonist activity of PAMs by providing a binding site at the TM6s interface. In the absence of orthosteric agonist, most probably a fraction of the molecules adopts this TM6s interface state, as observed for the mGlu2 receptor that constantly oscillates between the inactive and active conformation (Cao et al, 2021). This TM6s interface provides a binding site for the PAM that further stabilizes this active state.…”

Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

“…Based on structural (Doré et al, 2014;Du et al, 2021;Seven et al, 2021;Wu et al, 2014) and mutagenesis (Farinha et al, 2015;Gregory & Conn, 2015;Lundström et al, 2011) studies, the primary mGluR allosteric binding sites were determined to be located within the 7TM domain bundles. Previous work examining allosteric modulation of mGluR conformational dynamics generally used ensemble methods and was focused on the dimeric rearrangement of either the 7TM domain (Gutzeit et al, 2019;Nasrallah et al, 2021) or the extracellular ligand-binding domain (Cao et al, 2021). While these studies of individual domains provide insights into how allosteric modulators affect mGluR structure and dynamics, they are not conducive for the broader fingerprinting of the modulator effect across multiple domains of the receptor.…”

Multi-parameter photon-by-photon hidden Markov modeling

“…Applying these methods is as simple as assigning an index to each photon stream. We include a supplementary Jupyter notebook using a developer version of FRETBursts 81 that accepts fluorescence anisotropy information from multi-parameter fluorescence detection, or from MFD coupled to pulsed interleaved excitation 34 , 43 , and demonstrate mpH 2 MM’s ability to disentangle fluorescence anisotropies on data kindly provided by Cao et al 82 . Values within the emission probability matrix can then be used as intensities to calculate all relevant ratiometric values.…”

“…We thank Gregor Hagelücken and Martin Peter from the Institute of Structural Biology (University of Bonn, GER) for providing YopO. We would like to thank Robert Quast and Emmanuel Margeat for insightful discussions regarding the implementation of mpH 2 MM for the analysis of 4-detector nsALEX measurements (2-color smFRET, with fluorescence anisotropies), based on their existing data 82 . We would also like to thank Demain Lieberman for his helpful discussion regarding implementation of H 2 MM code, and Bill Harris for his help in enabling the H2MM_C code to work on Windows and Linux.…”