Development of an Accessible Cell-free Assay for the Purpose of Studying the Structural Mechanisms Underlying GPCR Ligand Molecular Efficacy

Abstract

G protein coupled receptors, (GPCRs) constitute the largest family of mammalian cell surface receptors and the most frequently targeted protein family by modern pharmaceuticals. The emergence of numerous high-resolution structures for GPCRs has energized structure-based drug discovery efforts to design potent, selective ligands that modulate pathological signaling (Roth, Irwin, and Shoichet 2017). However, GPCR signaling involves the serial amplification of ligand-receptor interactions, leading to non-linear downstream responses that do not correlate with receptor-level effects. Meanwhile, the ‘intrinsic efficacy’ parameter (Stephenson 1956; Kenakin 2014) incorporated by classical pharmacological models to capture system-independent effects of GPCR ligands remains elusive due to limitations in quantitative receptor-level information access. Recently, an extensive single molecule FRET based study has characterized a ligand ‘molecular efficacy’ parameter that captures the influence of ligand-dependent receptor conformation on G protein activation (Gregorio et al. 2017). Nonetheless, current efforts to elucidate the underlying mechanisms that dictate GPCR ligand molecular efficacy are limited by the requirement of extensive purification of receptor and G proteins to homogeneity. Hence, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. To bridge this knowledge gap, and circumvent the need for extensive purification protocols, we first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C-terminus (G-peptide), a well-known structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into Giant Plasma Membrane Vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor-G protein interactions. We demonstrate that GPMV incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology by combining v-SPASM sensor measurements with molecular dynamics (MD) simulations, we further elucidate a two-stage receptor activation mechanism, wherein receptor-G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.