1. Structural Biology and Molecular Biophysics
Download icon

Structural basis for mammalian nucleotide sugar transport

  1. Shivani Ahuja
  2. Matthew R Whorton  Is a corresponding author
  1. Oregon Health and Science University, United States
Research Article
  • Cited 12
  • Views 3,226
  • Annotations
Cite this article as: eLife 2019;8:e45221 doi: 10.7554/eLife.45221

Abstract

Nucleotide-sugar transporters (NSTs) are critical components of the cellular glycosylation machinery. They transport nucleotide-sugar conjugates into the Golgi lumen, where they are used for the glycosylation of proteins and lipids, and they then subsequently transport the nucleotide monophosphate byproduct back to the cytoplasm. Dysregulation of human NSTs causes several debilitating diseases, and NSTs are virulence factors for many pathogens. Here we present the first crystal structures of a mammalian NST, the mouse CMP-sialic acid transporter (mCST), in complex with its physiological substrates CMP and CMP-sialic acid. Detailed visualization of extensive protein-substrate interactions explains the mechanisms governing substrate selectivity. Further structural analysis of mCST's unique lumen-facing partially-occluded conformation, coupled with the characterization of substrate-induced quenching of mCST's intrinsic tryptophan fluorescence, reveals the concerted conformational transitions that occur during substrate transport. These results provide a framework for understanding the effects of disease-causing mutations and the mechanisms of this diverse family of transporters.

Data availability

Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with entries 6OH2, 6OH3, and 6OH4.

The following data sets were generated

Article and author information

Author details

  1. Shivani Ahuja

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Matthew R Whorton

    Vollum Institute, Oregon Health and Science University, Portland, United States
    For correspondence
    whorton@ohsu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9915-7467

Funding

Oregon Health and Science University

  • Shivani Ahuja
  • Matthew R Whorton

National Institutes of Health (R01GM130909)

  • Shivani Ahuja
  • Matthew R Whorton

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

Publication history

  1. Received: January 17, 2019
  2. Accepted: April 13, 2019
  3. Accepted Manuscript published: April 15, 2019 (version 1)
  4. Version of Record published: May 9, 2019 (version 2)
  5. Version of Record updated: May 15, 2019 (version 3)

Copyright

© 2019, Ahuja & Whorton

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

  • 3,226
    Page views
  • 537
    Downloads
  • 12
    Citations

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

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)

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

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

Further reading

    1. Physics of Living Systems
    2. Structural Biology and Molecular Biophysics
    Valentin Dunsing et al.
    Tools and Resources Updated

    Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.

    1. Structural Biology and Molecular Biophysics
    Abhishek Mazumder et al.
    Research Article Updated

    Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPo). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyse RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, ‘bind-unwind-load-and-lock’, model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.