Structural basis of endogenous lipid recognition and G protein selectivity in GPR119

Problem
G protein-coupled receptors (GPCRs) frequently engage multiple intracellular transducers, yet the structural mechanisms by which endogenous ligands dictate G protein selectivity remain poorly understood. GPR119, a lipid-activated receptor critical for glucose homeostasis and a therapeutic target for type 2 diabetes and obesity, is known to couple primarily to Gs but also to Gq and Gi pathways. While high-resolution structures of GPR119 bound to synthetic agonists in complex with Gs exist, the molecular basis for its recognition of its most potent endogenous agonist, oleoylethanolamide (OEA), and the structural determinants governing its coupling to distinct G protein subtypes (Gs vs. Gq) under physiological conditions have remained elusive.

Methodology
The authors employed a multidisciplinary approach combining structural biology, computational modeling, and functional assays:

• Protein Engineering and Expression: They engineered a human wild-type GPR119 construct with N-terminal tags (HA, FLAG, His, bRIL) and a C-terminal eGFP. To stabilize the complexes, they co-expressed GPR119 with either a dominant-negative Gαs (DNGαs) or an engineered miniGαq, along with Gβ1γ2 subunits. The Gs complex was further stabilized with nanobody 35 (Nb35).
• Cryo-Electron Microscopy (Cryo-EM): Single-particle cryo-EM was used to determine the structures of OEA-bound GPR119 in complex with Gs-Nb35 and Gq at resolutions of 2.72 Å and 3.05 Å, respectively.
• Molecular Dynamics (MD) Simulations: Simulations were performed on both the engineered constructs and models where these were replaced with wild-type sequences to validate stability and analyze conformational dynamics.
• Functional Assays: BRET-based ONE-GO assays and HTRF IP-ONE assays were utilized to measure Gs and Gq signaling activation, respectively, in HEK293T cells. Site-directed mutagenesis was performed on key residues identified in the structural analysis to assess their impact on ligand binding and G protein coupling.

Key Contributions and Results

1. Structural Basis of OEA Recognition: The study presents the first cryo-EM structures of GPR119 bound to its endogenous agonist OEA in complex with both Gs and Gq. OEA occupies a deeply buried orthosteric pocket formed by transmembrane helices (TMs) 2, 3, 5, 6, 7, and ECL2.
2. Distinct Ligand Conformations: Although the overall receptor architecture is similar between the two states (RMSD = 0.6 Å), OEA adopts distinct conformations. In the Gs-coupled state, OEA penetrates approximately 2.1 Å deeper into the receptor core compared to the Gq-coupled state. This deeper insertion in the Gs complex is facilitated by a specific interaction between the OEA C9-C10 double bond and the conserved toggle switch residue W2386.48, as well as an amide–π interaction between the OEA head group and W2657.39. In the Gq complex, W2657.39 adopts a flipped conformation, shifting the OEA head group toward the extracellular side and disrupting a specific hydrogen bond network (R2627.36–Q652.64) seen in the Gs state.
3. TM5 as a Determinant of Selectivity: A major structural difference identified is the conformation of Transmembrane Helix 5 (TM5). In the Gs complex, TM5 is extended by approximately three helical turns (residues 199–210), forming extensive interactions with the Ras-like domain of Gαs. This extended region is disordered in the Gq complex. Functional assays confirmed that residues within this extended TM5 region, specifically I1995.73 and K2015.75, are critical for Gs activation but have minimal impact on Gq signaling. A double mutant (I1995.73A/K2015.75A) significantly impaired Gs signaling while preserving Gq activity, identifying TM5 as a key structural determinant for G protein subtype selectivity.
4. Interface Comparisons: The study details shared and unique interactions at the receptor-G protein interface. While both complexes share a conserved insertion of the Gα C-terminal α5 helix into the intracellular cavity, the Gs complex exhibits a more extensive interaction network involving intracellular loops (ICL1 and ICL2) and the α5 helix compared to Gq. Mutagenesis of ICL residues (e.g., K35ICL1) revealed that Gq coupling is more sensitive to single-residue substitutions than Gs coupling, likely due to a sparser interaction network.
5. Comparative Analysis: Comparisons with other class A GPCRs (H2R, mTAAR1, FFAR4, CCK1R) suggest that while the specific residues governing selectivity are not conserved, the conformational state of the TM5–ICL3–TM6 region appears to be a recurring structural feature influencing G protein coupling across the family.

Significance
The paper claims that these findings elucidate the structural determinants underlying multi-transducer coupling of GPR119 driven by an endogenous lipid agonist. By revealing how subtle differences in ligand positioning propagate to the intracellular interface and how the extension of TM5 acts as a switch for Gs selectivity, the study provides a mechanistic framework for understanding G protein selectivity. The authors state that these results establish a structural foundation for the rational design of pathway-selective therapeutics targeting metabolic disorders such as type 2 diabetes and obesity, potentially enabling the development of ligands that can bias signaling toward beneficial pathways (e.g., Gs-mediated insulin secretion) or modulate specific metabolic outcomes.