Fig. 5: Modulation of biophysical and allosteric properties of GPCRs by directed evolution in mammalian cells.

GPCRs sample a multitude of conformational states that are allosterically modulated by ligand and G protein binding. Introduction of an uncoupling mutation (red star) leads to disruption of the signal transmission from the ligand binding pocket to the G protein interface (left path). Thus, in subsequent directed evolution in the absence of G protein or when its binding was prevented by a mutation already stabilizing the inactive state, preferentially mutations that further stabilize the receptor in an inactive state (grey) were selected, leading to a rigid and stable conformation. If receptor function is retained at the beginning of selection, the cellular environment, which contains G proteins, dictates the selection outcome. Mutations promoting allosteric coupling between the agonist-occupied conformation of the ligand binding pocket and the active-state (AS) conformation of the G protein binding interface (blue) were selected. In some cases, mutations were enriched that promote a strong allosteric coupling of the active-state conformation, resulting in constitutive receptor activity (green). Figure modified after Nygaard et al.⁶⁰.