Structural mechanisms of phospholipid activation of the human TPC2 channel

  1. Ji She
  2. Weizhong Zeng
  3. Jiangtao Guo
  4. Qingfeng Chen
  5. Xiaochen Bai  Is a corresponding author
  6. Youxing Jiang  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. Zhejiang University School of Medicine, China

Abstract

Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-activated, Na+ selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P2-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P2 binds at the first 6-TM and activates the channel - independently of the membrane potential - by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.

Data availability

The cryo-EM density maps of the human TPC2 have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-0478 for the apo state, EMD-0479 for the PI(3,5)P2-bound closed state and EMD-0477 for the PI(3,5)P2-bound open state. Atomic coordinates have been deposited in the Protein Data Bank under accession numbers 6NQ1 for the apo state, 6NQ2 for the PI(3,5)P2-bound closed state and 6NQ0 for the PI(3,5)P2-bound open state.

The following data sets were generated

Article and author information

Author details

  1. Ji She

    Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7006-6230
  2. Weizhong Zeng

    Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Jiangtao Guo

    Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Qingfeng Chen

    Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Xiaochen Bai

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    Xiaochen.Bai@UTSouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.
  6. Youxing Jiang

    Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    youxing.jiang@utsouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1874-0504

Funding

Howard Hughes Medical Institute

  • Youxing Jiang

National Institute of General Medical Sciences (GM079179)

  • Youxing Jiang

Welch Foundation (I-1578)

  • Youxing Jiang

Cancer Prevention and Research Initiative of Texas

  • Xiaochen Bai

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Baron Chanda, University of Wisconsin-Madison, United States

Version history

  1. Received: January 16, 2019
  2. Accepted: March 7, 2019
  3. Accepted Manuscript published: March 12, 2019 (version 1)
  4. Version of Record published: March 19, 2019 (version 2)
  5. Version of Record updated: July 17, 2019 (version 3)

Copyright

© 2019, She et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 4,248
    Page views
  • 768
    Downloads
  • 85
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Ji She
  2. Weizhong Zeng
  3. Jiangtao Guo
  4. Qingfeng Chen
  5. Xiaochen Bai
  6. Youxing Jiang
(2019)
Structural mechanisms of phospholipid activation of the human TPC2 channel
eLife 8:e45222.
https://doi.org/10.7554/eLife.45222

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Daniel Muñoz-Reyes, Levi J McClelland ... Maria Jose Sanchez-Barrena
    Research Article

    The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Bronwyn A Lucas, Benjamin A Himes, Nikolaus Grigorieff
    Research Advance

    Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.