Basal and ligand-induced conformational ensembles in G protein-coupled receptor signaling [electronic resource]
Keywords: Chemical and Systems Biology.

G protein coupled receptor proteins (GPCRs) couple extracellular soluble ligand binding to intracellular signaling pathway activation. GPCRs are excellent drug targets due to their positioning at the plasma membrane upstream of signaling cascades, and due to their ability to respond to ligand binding with a range of signaling outputs. Crystallography has revealed the structure of drugs bound to the inactive and active state GPCRs at high resolution; each crystal structure represents a single, low-energy GPCR conformation. Crystal structures have also defined the canonical activation mechanism for GPCRs as an opening up of the intracellular surface; for Family A GPCRs, the intracellular portion of transmembrane helix 6 (TM6) undergoes the largest conformational change upon ligand binding. Here I present the design and development of two novel fusion proteins for GPCR crystallography. Crystal structures of the M3 muscarinic receptor fused to both novel crystallization aids improved the resolution of the overall structure and extended the view of residues comprising TM6. In addition to the states seen by crystallography, recent spectroscopy studies revealed that GPCRs sample a wide variety of conformations. This raises the question of when and whether high-energy (non-crystallographic) conformations are relevant for GPCR function. In this work, I both employ and validate a high-pressure electron paramagnetic resonance (EPR) spectroscopy technique to understand the basal and ligand activation of an archetypal GPCR, the Beta-2 adrenergic receptor (β2AR). The studies demonstrate a pre-existing equilibrium between inactive and active receptor states, providing a structure-based explanation for the observed phenomenon of basal activity. Clinically, inverse agonists are used to inhibit basal activity of GPCRs, and these high-pressure studies also reveal a structural mechanism for inverse agonists: they inhibit the outward movement of TM6. Studies of β2AR endogenous and synthetic agonists under ambient and pressurized conditions reveal a distinct conformational profile for each agonist. These conformational differences may be responsible for different ligand efficacies towards two signaling pathways downstream of GPCRs: arrestin-mediated versus G-protein-mediated signaling. Overall, these studies provide further support for a general mechanism for GPCR activation, conformational selection. The ability to fine-tune drug efficacy is the next frontier in GPCR drug discovery. To this end, I contributed to a collaborative drug discovery project to find novel β2AR allosteric ligands by docking to a hypothetical allosteric binding site, at a distinct location from where endogenous ligands bind. We built upon the principles of conformational selection, searching for negative modulators by docking to the inactive crystal structure, and finding positive modulators from active-state structure docking. Some of the new allosteric ligands discovered have signal bias properties, targeting arrestin over G-protein coupling. Though the underlying mechanisms of ligand bias are difficult to assess by most physiologic and biophysical methods, EPR spectroscopy revealed subtle differences in the conformational ensemble, which may correspond to arrestin versus G protein-specific drug effects.