1. Structural Biology and Molecular Biophysics

Cryo-EM structures of human ZnT8 in both outward- and inward-facing conformations

  1. Jing Xue
  2. Tian Xie
  3. Weizhong Zeng
  4. Youxing Jiang  Is a corresponding author
  5. Xiao-chen Bai  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
https://doi.org/10.7554/eLife.58823
Share this article
Cite this article
  1. Jing Xue
  2. Tian Xie
  3. Weizhong Zeng
  4. Youxing Jiang
  5. Xiao-chen Bai
(2020)
Cryo-EM structures of human ZnT8 in both outward- and inward-facing conformations
eLife 9:e58823.
https://doi.org/10.7554/eLife.58823
  2. Download BibTeX
  3. Download .RIS

Abstract

ZnT8 is a Zn2+/H+ antiporter that belongs to SLC30 family and plays an essential role in regulating Zn2+ accumulation in the insulin secretory granules of pancreatic β cells. Dysfunction of ZnT8 is associated with both type 1 and 2 diabetes. However, the Zn2+/H+ exchange mechanism of ZnT8 remains unclear due to the lack of high-resolution structures. Here, we report the cryo-EM structures of human ZnT8 (HsZnT8) in both outward- and inward-facing conformations. HsZnT8 forms a dimeric structure with four Zn2+ binding sites within each subunit: a highly conserved primary site in transmembrane domain (TMD) housing the Zn2+ substrate; an interfacial site between TMD and C-terminal domain (CTD) that modulates the Zn2+ transport activity of HsZnT8; and two adjacent sites buried in the cytosolic domain and chelated by conserved residues from CTD and the His-Cys-His (HCH) motif from the N-terminal segment of the neighboring subunit. A comparison of the outward- and inward-facing structures reveals that the TMD of each HsZnT8 subunit undergoes a large structural rearrangement, allowing for alternating access to the primary Zn2+ site during the transport cycle. Collectively, our studies provide the structural insights into the Zn2+/H+ exchange mechanism of HsZnT8.

Data availability

The Cryo-EM maps of HsZnT8 determined in three different conditions will have been deposited in the Electron Microscopy Data Bank and. The corresponding atomic coordinates will be deposited to the RCSB Protein Data Bank, with the entry ID: EMD-22285 and PDB 6XPD for the structure of HsZnT8-DM, EMD-22286 and PDB 6XPE for the structure of HsZnT8-WT in the presence of zinc, and EMD-22287 and PDB 6XPF for the structure of HsZnT8-WT in the absence of zinc. Source data files have been provided for Figure 2h and 2i.

The following previously published data sets were used

Article and author information

Author details

  1. Jing Xue

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

  2. Tian Xie

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

  3. Weizhong Zeng

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

  4. 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

  5. Xiao-chen 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.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4234-5686

Funding

National Institute of General Medical Sciences (R01GM136976)

  • Xiao-chen Bai

Welch Foundation (I-1944)

  • Xiao-chen Bai

Cancer Prevention and Research Institute of Texas (RP160082)

  • Xiao-chen Bai

Howard Hughes Medical Institute

  • Youxing Jiang

National Institute of General Medical Sciences (GM079179)

  • Youxing Jiang

Welch Foundation (I-1578)

  • Youxing Jiang

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

Reviewing Editor

  1. Olga Boudker, Weill Cornell Medicine, United States

Version history

  1. Received: May 12, 2020
  2. Accepted: July 28, 2020
  3. Accepted Manuscript published: July 29, 2020 (version 1)
  4. Version of Record published: August 14, 2020 (version 2)

Copyright

© 2020, Xue 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

  • 4,987
    views
  • 917
    downloads
  • 50
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Jing Xue
  2. Tian Xie
  3. Weizhong Zeng
  4. Youxing Jiang
  5. Xiao-chen Bai
(2020)
Cryo-EM structures of human ZnT8 in both outward- and inward-facing conformations
eLife 9:e58823.
https://doi.org/10.7554/eLife.58823

Share this article

https://doi.org/10.7554/eLife.58823

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA

    Claudia D Consalvo, Adedeji M Aderounmu ... Brenda L Bass
    Research Article

    Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1’s helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors

    Damien M Rasmussen, Manny M Semonis ... Nicholas M Levinson
    Research Article

    The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

    1. Structural Biology and Molecular Biophysics

    Some mechanistic underpinnings of molecular adaptations of SARS-COV-2 spike protein by integrating candidate adaptive polymorphisms with protein dynamics

    Nicholas James Ose, Paul Campitelli ... Sefika Banu Ozkan
    Research Article

    We integrate evolutionary predictions based on the neutral theory of molecular evolution with protein dynamics to generate mechanistic insight into the molecular adaptations of the SARS-COV-2 spike (S) protein. With this approach, we first identified candidate adaptive polymorphisms (CAPs) of the SARS-CoV-2 S protein and assessed the impact of these CAPs through dynamics analysis. Not only have we found that CAPs frequently overlap with well-known functional sites, but also, using several different dynamics-based metrics, we reveal the critical allosteric interplay between SARS-CoV-2 CAPs and the S protein binding sites with the human ACE2 (hACE2) protein. CAPs interact far differently with the hACE2 binding site residues in the open conformation of the S protein compared to the closed form. In particular, the CAP sites control the dynamics of binding residues in the open state, suggesting an allosteric control of hACE2 binding. We also explored the characteristic mutations of different SARS-CoV-2 strains to find dynamic hallmarks and potential effects of future mutations. Our analyses reveal that Delta strain-specific variants have non-additive (i.e., epistatic) interactions with CAP sites, whereas the less pathogenic Omicron strains have mostly additive mutations. Finally, our dynamics-based analysis suggests that the novel mutations observed in the Omicron strain epistatically interact with the CAP sites to help escape antibody binding.