Observation of Structural Changes on Activation of the NTS1 G-Protein-Coupled Receptor on DNA-Templated Protein Arrays by cryo-EM

We report the use of self-assembled DNA templates to create dense, orientationally disordered protein arrays that are optimized for single-particle cryo-EM. The nanostructure templates dramatically simplify data collection and have allowed us to obtain the first structures of a wild-type, neuropeptide-binding GPCR, the rat neurotensin receptor type 1 (NTS1), in both its ligand-free and liganded forms. Resolution better than 7A allows clear identification of the 7 trans-membrane (TM) alpha-helices. Comparison of the structures provides the first direct observations and measurements of helix excursions during the conformational changes associated with activation of a ligand-binding GPCR. Conformational changes in the TM helices are observed upon ligand binding, namely shifts in TM1 and TM2 at the extracellular side of the membrane and pronounced shifts in TM5 and TM6 on the intracellular, G-protein interacting side that are the hallmark of the GPCR-activated state. Our results suggest a mechanism for ligand activation of a class A GPCR.This is the highest resolution achieved to date by single-particle cryo-EM of a membrane protein, and NTS1 (43kDa) is an order of magnitude smaller than the nearest similar existing reconstruction of an asymmetric protein at comparable resolution. Our results suggest that the use of DNA-templated protein arrays has the potential to make high-resolution structure determination for small, asymmetric and hard-to-crystallize proteins routinely achievable.