Impact of critical-site mutations in the free fatty acid receptor 4 on the allosteric kinetics of endogenous fatty acid-receptor interactions.

BACKGROUND Free Fatty Acid Receptor 4 (FFAR4/GPR120) is a lipid-sensing GPCR, yet quantitative evidence linking binding-pocket residues to endogenous fatty-acid selectivity and coupling behavior remains limited. In particular, how Arg99, Trp198 and Trp207 cooperatively shape ligand-class dependence (saturated vs unsaturated; short-vs long-chain) has not been systematically quantified using a unified experimental metric.

RESULTS An AuNP-assisted, HRP/Thionine-amplified electrochemical GPR120 receptor sensor was constructed and stepwise assembly was verified by cyclic voltammetry. Steady-state chronoamperometry was performed for wild-type GPR120 and engineered variants with 14 natural fatty acids, and plateau currents were converted to ΔI(%) (mean ± SD, n = 3 independent electrodes). An operational coupling parameter Ka (ligand concentration producing a half-maximal steady-state response, ΔI%, plateau value) was extracted by double-reciprocal linearization with high linearity (R2 = 0.931-0.997, most ≥0.95). For wild-type GPR120, Ka values ranged from 2.87 × 10-17 to 1.78 × 10-13 M, revealing chain-length dependence and ligand-class differences in steady-state coupling. Mutations induced pronounced ligand-dependent shifts, exemplified by butyric acid (WT 2.73 × 10-15 → mut1 4.05 × 10-13 M), linoleic acid (WT 3.80 × 10-15 → mut2 4.24 × 10-16 M), and palmitic acid (WT 6.87 × 10-15 → mut3 2.10 × 10-13 M). Molecular docking (AutoDock Vina; exhaustiveness = 8; 9 output poses; grid-box settings in Table 1) and 100-ns molecular dynamics simulations (GROMACS 2022.1, CHARMM36 m/TIP3P; 2 fs; 300 K; 1 bar; PME; 1.0 nm cutoff) supported the Ka trends by revealing ligand-class-dependent pocket accommodation and mutation-dependent changes in interaction networks and stability signatures. Quantitatively, backbone RMSD mainly fluctuated within 0.5-1.5 nm, protein-ligand hydrogen bonds were typically ∼1-4, and radius of gyration variations were <0.05 nm, indicating overall stable folds with mutation-dependent local pocket rearrangements.

SIGNIFICANCE This work provides a practical electrochemical-computational framework for residue-resolved profiling of GPR120-fatty acid coupling behavior using Ka as a unified quantitative metric. Experimentally, the workflow yielded 56 quantitative Ka values (14 fatty acids × WT and mut1-3) with robust extraction quality (R2 = 0.931-0.997). Class-stratified analysis using ΔlogKa = log10[Ka(mut)/Ka(WT)] revealed residue-dependent trends in coupling behavior. In particular, mut1 and mut2 showed different shifts between saturated and unsaturated fatty acids (mut1: SFA +0.55 ± 0.75 vs UFA -0.21 ± 0.47; mut2: SFA +0.50 ± 0.54 vs UFA -0.50 ± 0.40), consistent with an important contribution to headgroup recognition. The Trp198/Trp207 variant generally exhibited higher Ka values for many tested fatty acids (e.g., palmitic acid 6.87 × 10-15 to 2.10 × 10-13 M), suggesting altered hydrophobic/aromatic packing associated with ligand-class-dependent differences across saturated/unsaturated and short-/long-chain fatty acids.