**Background:** ATP-binding cassette (ABC) transporters are membrane proteins that translocate diverse molecules across biological membranes. Plant genomes are exceptionally rich in ABCG transporters, which transport lipids, phytohormones, and specialized metabolites. MtABCG46 from Medicago truncatula selectively transports 4-coumarate and liquiritigenin, but not structurally similar phenylpropanoids like naringenin, isoliquiritigenin, or 7,4′-dihydroxyflavone. The molecular basis for this selectivity was previously unknown.
**Methods:** The authors used AlphaFold2 v2.1.0 to predict the 3D structure of MtABCG46. Molecular dynamics (MD) simulations were performed using Amber20 with ff19SB force field, POPC lipid bilayer, and OPC water model. Access path detection used CAVER v3.0 with a 0.9 Å probe radius. Ligand migration was studied with CaverDock v1.1. Phylogenetic analysis used 1839 full-size ABCG sequences from the 1KP project, analyzed with ConSurf and Gremlin servers. For experimental validation, MtABCG46 variants (WT, F562L, F562Y, F562A) with N-terminal GFP tags were expressed in Nicotiana tabacum BY2 suspension cells via Agrobacterium tumefaciens transformation. Plasma membrane vesicles were isolated by aqueous two-phase partitioning. Transport assays used rapid filtration with 750 μM phenolic compounds, 4 mM ATP, 3 min incubation at 24°C, analyzed by LC/ESI/MS. Statistical analyses used GraphPad Prism v9.0 with Kruskal-Wallis test and post hoc Dunn's multiple comparison test.
**Key Results:** The AlphaFold2 model of MtABCG46 revealed an occluded central cavity, contrasting with the open cavity of ScPDR5. MD simulations identified a transient access path from the intracellular region to the central cavity, open for ~9% of simulation time in WT, with an average bottleneck radius of 0.97 ± 0.06 Å. Phylogenetic analysis showed residue F562 in TMD helix 2 corresponds to F431 in human ABCG2, a residue important for ligand recognition. In seed plants, variability at this position (phenylalanine, tyrosine, leucine, isoleucine) was significantly higher than in non-seed plants. Experimental transport assays showed WT MtABCG46 transported liquiritigenin and 4-coumarate but not isoliquiritigenin or 7,4′-dihydroxyflavone. F562Y and F562A substitutions abolished transport of both substrates. F562L retained liquiritigenin transport similar to WT but lost 4-coumarate transport. Competition assays showed 4-coumarate, isoliquiritigenin, and 7,4′-dihydroxyflavone all competed with liquiritigenin transport, with increased susceptibility in F562L. Umbrella sampling simulations showed the opening process had 1.2, 9.4, and 11.6 kcal mol⁻¹ higher energetic costs in F562L, F562Y, and F562A, respectively, compared to WT at RMSD ~1.5 Å. In IF-open state simulations, WT showed access paths open for ≥31% of simulation time with bottleneck radii up to 1.7 Å, while F562L showed paths open for ≥12% with radii up to 1.4 Å. CaverDock calculations showed nearly 100% successful migration for 4-coumarate in WT IF-open state, ~60% for liquiritigenin and 7,4′-dihydroxyflavone, and only ~30% for isoliquiritigenin. In F562L, 4-coumarate retained about half its efficiency, while all bulkier compounds showed much lower success rates.
**Clinical Implications:** While this study focuses on plant biology, the mechanistic insights into ABCG transporter selectivity have broader implications. ABCG transporters are clinically important due to their role in multidrug resistance in fungi and humans. Understanding how restriction of access to the central cavity contributes to substrate selectivity provides a framework for engineering or inhibiting these transporters. The identification of F562 as a critical residue for access path architecture and the demonstration that single residue substitutions can dramatically alter selectivity profiles may inform efforts to modulate human ABCG2 (which shares the corresponding F431 residue) in drug resistance contexts. The combined computational-experimental approach also demonstrates a strategy for studying membrane transporters that are difficult to crystallize.