Receptor hyper-flexibility of human bitter receptor TAS2R4 revealed by cryo-EM structures in apo and tripeptide-bound states

Human bitter taste receptors (TAS2Rs) represent a unique class of G protein-coupled receptors (GPCRs) responsible for detecting a vast array of chemically diverse bitter compounds 1 , 2 . Unlike the more specialized sweet or umami receptors, ~26 members of the human TAS2R (hTAS2R) family must balance broad-spectrum recognition with fine-tuned signal transduction 3 . Among these, TAS2R4 stands out as an intermediately tuned receptor capable of responding to structurally distinct agonists, including various short-chain bitter peptides found in fermented foods (Supplementary Fig. S1 ) 4 , 5 . Despite the recent publications of TAS2R structural data 6 , 7 , 8 , the dynamic transition from an apo receptor to an active signaling complex remains largely elusive. Here, we present the cryo-electron microscopy (cryo-EM) structures of TAS2R4 in complex with mini-G protein gustducin (miniG gust ) in both apo and tripeptide 1 WWW 3 (3W)-bound states (Fig. 1a–d ). Our findings reveal a remarkable degree of receptor hyper-flexibility and a distinct activation mechanism that challenges current paradigms of class T GPCR signaling. Fig. 1: Cryo-EM structures of human bitter receptor TAS2R4 in apo and tripeptide-bound states. The alternative text for this image may have been generated using AI. Full size image a Cryo-EM density map of 3W–TAS2R4–miniGα gust complex. The density for 3W is clearly resolved in the ligand-binding pocket. b Cartoon representation of TAS2R4–miniGα gust complex in the 3W-bound state, showing the transmembrane domain of TAS2R4 (orange), miniGα gust (green), Gβ 3 (blue), Gγ 13 (brown), and scFv16 (gray), with 3W represented in purple. c Cryo-EM density map of apo–TAS2R4–miniGα gust complex. d Cartoon representation of apo–TAS2R4–miniGα gust complex, showing the transmembrane domain of TAS2R4 (cyan), miniGα gust (green), Gβ 3 (blue), Gγ 13 (brown), and scFv16 (gray). e The overall illustrations of the interaction of 3W with TAS2R4, highlighting the key TMs (TM1, TM2, TM3, TM6, and TM7) involved in forming the orthosteric site (top left), with the key residues shown as sticks (top right); and close-up illustrations of key interactions in the binding pocket, highlighting the bottom (botton left), wall (botton middle), and extracellular edge (botton right) of the orthosteric site. f Structural comparison of TAS2R4 in the apo (cyan) and 3W-bound (orange) states. Left: side view of the superimposed structures. Right: views from the extracellular and intracellular perspectives, illustrating the movements of TMs (TM1–TM7) between the apo and 3W-bound states. g Key stabilizing interactions in the apo state. Residues involved in hydrogen bonding are shown with their side chains in blue. h Structural rearrangement of the residues shown in g after 3W binding. i Movement of TM6 in the 3W-bound state relative to the apo state. j Superimposed structures of apo and 3W-bound TAS2R4–miniGα gust complexes emphasize the lateral (2.5 Å) and rotational (18°) movements of the α5 helix of miniGα gust upon ligand binding. k , l Interaction diagrams between the α5 helix of miniGα gust and TAS2R4 in the apo ( k ) and 3W-bound ( l ) states. Left: interactions with TM3 and TM6. Middle: simplified intracellular view illustrating the rearrangement of key interactions, where blue dashed lines represent polar interactions and brown dashed lines represent hydrophobic interactions. Right: interactions with TM2 and TM7. m Overall view of the interactions between TAS2R4 ICLs (ICL1, ICL2, ICL3) and G protein subunits (miniGα gust and Gβ) in the 3W-bound state. n Close-up views of specific interactions between TAS2R4 ICLs (ICL1, ICL2, ICL3) and G protein subunits (miniGα gust and Gβ) in the 3W-bound state. o Conformations of key residues involved in TAS2R4 activation in the apo and 3W-bound states. Upon 3W entering the binding pocket, Y250 6.56 undergoes a significant rotation, swinging outward to accommodate the ligand. Conformational changes of the same residues upon 3W binding are shown.