Structural basis for the transport mechanism and cholesterol modulation of the human proline transporter

L-proline, an amino acid, acts as a potent neuromodulator in the central nervous system, with its synaptic concentrations finely regulated by the high-affinity L-proline transporter (PROT). Dysregulation of PROT function is implicated in neuropsychiatric disorders, yet its regulatory mechanisms remain poorly characterized. Here, we present high-resolution cryo-electron microscopy structures of human PROT in L-proline-free consensus and L-proline-bound states. The L-proline-bound structure reveals an occluded conformation, detailing the coordination of substrate and ions. Strikingly, our L-proline-free consensus structures capture an inward-open conformation with a cholesterol molecule bound directly within the substrate-binding site. Transport kinetics studies demonstrate that partial cholesterol depletion increases PROT’s apparent affinity for L-proline, confirming that cholesterol inhibits L-proline uptake. Furthermore, we identify a conformationally sensitive site (CHOL1’) present only in the inward-open conformation. This work elucidates PROT’s transport cycle and demonstrates how cholesterol directly modulates neurotransmitter uptake, offering potential therapeutic strategies for brain disorders. Synaptic L-proline levels are regulated by its transporter (PROT). Here, authors present cryo-EM structures of human PROT, revealing substrate recognition and a direct inhibitory mechanism via cholesterol binding in the substrate-binding pocket.