Differences between G‐Protein‐Stabilized Agonist–GPCR Complexes and their Nanobody‐Stabilized Equivalents

Protein nanobodies have been used successfully as surrogates for unstable G-proteins in order to crystallize G-protein coupled receptors (GPCRs) in their active states. We have now used molecular dynamics (MD) simulations including metadynamics enhanced sampling to investigate the similarities and differences between GPCR-agonist ternary complexes with the -subunits of the appropriate G-proteins and with the protein nanobodies (intracellular binding partners, IBPs) used for crystallization. In two of three receptors considered, the agonist binding mode differs significantly between the two alternative ternary complexes. The ternary complex model of GPCR-activation entails enhancement of ligand binding by bound IBPs: Our results show IBP-specific changes that can alter the agonist binding modes and thus also the criteria for designing GPCR agonists