Structural basis of glucosinolate recognition and transport by plant GTR1

Abstract Glucosinolates (GLSs) play crucial roles in plant defense against herbivores. GTR1 facilitates the high-affinity transport of GLSs through a proton-dependent process. However, the molecular mechanism underlying GLS recognition and transport by GTR1 remains largely unknown. Here, we present four cryo-EM structures of Arabidopsis GTR1 in distinct states, namely, the outward-apo, inward-apo, 4MTB-bound and 3IMG-bound forms, revealing the structural basis for GLS and proton cotransport by GTR1. GTR1 consists of an MFS-like transmembrane domain and an intracellular domain (ICD). The ICD plays an essential role in GTR1 function by interacting with the gating helix, transmembrane helix 7. GLSs are recognized by the central cavity residues and directly interact with the conserved E 1 X 1 X 2 E 2 K motif. Our structural and functional analyses demonstrated that the E 1 X 1 X 2 E 2 K motif and Glu513 determine the proton coupling of GTR1. This study provides mechanistic insights into how GTR1 transports GLSs, which could aid in improving crop quality and enhancing resistance to herbivory.