Abstract
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.
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Acknowledgements
We gratefully acknowledge all members of the Wu laboratory for their invaluable scientific discussions and technical assistance. The authors appreciate the AKTA purification platform provided by the labs of Pingping Li and Bing Cui, the experimental equipment provided by the lab of Rui Wang, and the centrifuge experimental platform provided by the Biological Analysis Center at the Institute of Materia Medica of Chinese Academy of Medical Sciences. Cryo-EM data collection was supported by the Electron Microscopy Laboratory, Cryo-EM Platform of Peking University, and Shuimu BioSciences Ltd. with the assistance of Xuemei Li, Zhenxi Guo, Changdong Qin, Xia Pei, Xiaojuan Hui, and Guopeng Wang. We also thank Shuimu BioSciences for cryo-EM facility access and technical support during image acquisition. Part of the structural computation was also performed on the Computing Platform of the Center for Life Science and the High-performance Computing Platform of Peking University. J.-X. W. discloses support for the research of this work from the National Natural Science Foundation of China [32522046 and 32371266 to J.-X. W.], CAMS Innovation Fund for Medical Sciences (CIFMS) [2025-I2M-QN-005 to J.-X. W.], the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences [No. 2022-RC350-01 to J.-X. W.] and Scientific Research Innovation Capability Support Project for Young Faculty [ZYGXQNJSKYCXNLZCXM-B2 to J.-X. W.]. N.W., J.W., Y.Z., and X.M. declare no relevant funding.
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Zhou, Y., Wang, N., Wang, J. et al. Structural basis for the transport mechanism and cholesterol modulation of the human proline transporter. Nat Commun (2026). https://doi.org/10.1038/s41467-026-72780-3
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DOI: https://doi.org/10.1038/s41467-026-72780-3
