Structural basis of glucosinolate binding and transport by Arabidopsis thaliana GTRs

Plants rely on specialized metabolites as chemical defenses, and conversely, accumulation of defense molecules can compromise crop quality, underscoring the need to clarify how the distribution of such compounds is established. Although several transporter families have been implicated, the molecular basis of cargo recognition and translocation remains unclear. Here, we present three cryo-EM structures of a key member of the Nitrate and Peptide transporter Family (NPF), GTR1, which is essential for cellular import of glucosinolate (GLS) defense compounds in the Brassicales plant order. The structures capture distinct conformations consistent with an alternating-access mechanism to a vestibule between the N- and C-terminal bundles, with the latter remaining largely static. Combined with in vitro and in silico studies, we define the GLS-binding pocket and identify protonation events likely required for transport. These insights establish a framework for understanding the mechanistic principles and a foundation for dissecting cargo recognition of the NPF.