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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月6日

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  • Yue Zhou,
  • Ningqin Wang,
  • Jiaqi Wang,
  • Xinrui Meng,
  • Jing-Xiang Wu

摘要

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.