A key residue of the extracellular gate provides quality control contributing to ABCG substrate specificity

For G-type ATP-binding cassette (ABC) transporters, a hydrophobic "di-leucine motif" as part of a hydrophobic extracellular gate has been described to separate a large substrate-binding cavity from a smaller upper cavity and proposed to act as a valve controlling drug extrusion. Here, we show that an L704F mutation in the hydrophobic extracellular gate of Arabidopsis ABCG36/PDR8/PEN3 uncouples the export of the auxin precursor indole-3-butyric acid (IBA) from that of the defense compound camalexin (CLX). Molecular dynamics simulations reveal increased free energy for CLX translocation in ABCG36^L704F and reduced CLX contacts within the binding pocket proximal to the extracellular gate region. Mutation L704Y enables export of structurally related non-ABCG36 substrates, IAA, and indole, indicating allosteric communication between the extracellular gate and distant transport pathway regions. An evolutionary analysis identifies L704 as a Brassicaceae family-specific key residue of the extracellular gate that controls the identity of chemically similar substrates. In summary, our work supports the conclusion that L704 is a key residue of the extracellular gate that provides a final quality control contributing to ABCG substrate specificity, allowing for balance of growth-defense trade-offs.