Abstract
Free fatty acid receptor 2 (FFA2) is a G protein-coupled receptor (GPCR) that is a primary sensor for short-chain fatty acids produced by gut microbiota. Consequently, FFA2 is a promising drug target for immunometabolic disorders(1-4). Here we report cryogenic electronic microscopy structures of FFA2 in complex with two G proteins and three distinct classes of positive allosteric modulators (PAMs), and describe noncanonical activation mechanisms that involve conserved structural features of class A GPCRs. Two PAMs disrupt the E/DRY activation microswitch(5) and stabilize the conformation of intracellular loop 2 by binding to lipid-facing pockets near the cytoplasmic side of the receptor. By contrast, the third PAM promotes the separation of transmembrane helices 6 and 7 by interacting with transmembrane helix 6 at the receptor-lipid interface. Molecular dynamic simulations and mutagenesis experiments confirm these noncanonical activation mechanisms. Furthermore, we demonstrate the molecular basis for the G(i) versus G(q) bias, which is due to distinct conformations of intracellular loop 2 stabilized by different PAMs. These findings provide a framework for the design of tailored GPCR modulators, with implications that extend beyond FFA2 to the broader field of GPCR drug discovery.