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Cryo-EM structures of the human endolysosomal TRPML3 channel in three distinct states

Nature Structural & Molecular Biology volume 24, pages 11461154 (2017) | Download Citation

Abstract

TRPML3 channels are mainly localized to endolysosomes and play a critical role in the endocytic pathway. Their dysfunction causes deafness and pigmentation defects in mice. TRPML3 activity is inhibited by low endolysosomal pH. Here we present cryo-electron microscopy (cryo-EM) structures of human TRPML3 in the closed, agonist-activated, and low-pH-inhibited states, with resolutions of 4.06, 3.62, and 4.65 Å, respectively. The agonist ML-SA1 lodges between S5 and S6 and opens an S6 gate. A polycystin-mucolipin domain (PMD) forms a luminal cap. S1 extends into this cap, forming a 'gating rod' that connects directly to a luminal pore loop, which undergoes dramatic conformational changes in response to low pH. S2 extends intracellularly and interacts with several intracellular regions to form a 'gating knob'. These unique structural features, combined with the results of electrophysiological studies, indicate a new mechanism by which luminal pH and other physiological modulators such as PIP2 regulate TRPML3 by changing S1 and S2 conformations.

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Acknowledgements

This work was supported by the National Basic Research Program of China (grant 2014CB910301 to J.Y.), the National Institutes of Health (grant R01GM085234 to J.Y.), the National Natural Science Foundation of China (grant 31370821 to J.Y.; grant 31570730 to X.L.), the National Key Research and Development Program (grants 2016YFA0501102 and 2016YFA0501902 to X.L.), the Top Talents Program of Yunnan Province (grant 2011HA012 to J.Y.), the High-level Overseas Talents of Yunnan Province (J.Y.), the China Youth 1000-Talent Program of the State Council of China (X.L.), Beijing Advanced Innovation Center for Structural Biology (X.L.), and the Tsinghua-Peking Joint Center for Life Sciences (X.L.).

Author information

Author notes

    • Xiaoyuan Zhou
    • , Minghui Li
    •  & Deyuan Su

    These authors contributed equally to this work.

Affiliations

  1. Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China.

    • Xiaoyuan Zhou
    •  & Xueming Li
  2. Department of Biological Sciences, Columbia University, New York, New York, USA.

    • Minghui Li
    • , Deyuan Su
    •  & Jian Yang
  3. Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences, Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China.

    • Deyuan Su
    • , Huan Li
    •  & Jian Yang
  4. Department of Orthopedic Oncology, Shanghai Changzheng Hospital, The Second Military Medical University, Shanghai, China.

    • Qi Jia
  5. Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China.

    • Huan Li

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Contributions

M.L. and J.Y. conceived and initiated the project. X.Z., M.L., D.S., Q.J., H.L., X.L., and J.Y. designed the experiments, analyzed the results, and wrote the manuscript. M.L. performed all molecular biology and biochemical experiments and built the atomic models. X.Z. and X.L. performed all cryo-EM experiments, including data acquisition and processing. D.S., Q.J., and H.L. performed electrophysiology experiments. All authors contributed to manuscript discussion, preparation, and editing.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Xueming Li or Jian Yang.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6

  2. 2.

    Life Sciences Reporting Summary

    Original electrophysiological data for Figures 2 and 5

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    Supplementary Data Set 1

    Source data for Figures 2 and 5.

Videos

  1. 1.

    Conformational changes induced by the binding of ML-SA1

    ML-SA1 binding causes many movements. For example, when viewed from the side, S5 and S6 move outward and the S4-S5 linker moves downward by 2 to 4 Å, the pore-loop moves downward by 2 Å, and S6 undergoes a 27 degree counterclockwise rotation. The zoom-in view from the bottom shows the movement of I498 (in space-filling form) between the closed state and open state upon ML-SA1 binding and unbinding.

  2. 2.

    Conformational changes induced by pH changes

    The movie shows a morph between the pH 7.4 apo structure and the pH 4.8 apo structure. The channel is viewed first from the side (parallel to the membrane) and then from top down (perpendicular to the membrane).

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DOI

https://doi.org/10.1038/nsmb.3502

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