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

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

  • 3,449
    Page views
  • 568
    Downloads
  • 16
    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)

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. Shivani Ahuja
  2. Matthew R Whorton
(2019)
Structural basis for mammalian nucleotide sugar transport
eLife 8:e45221.
https://doi.org/10.7554/eLife.45221

Further reading

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics
    Claudia C Carcamo et al.
    Research Article Updated

    One-dimensional (1D) target search is a well-characterized phenomenon for many DNA-binding proteins but is poorly understood for chromatin remodelers. Herein, we characterize the 1D scanning properties of SWR1, a conserved yeast chromatin remodeler that performs histone exchange on +1 nucleosomes adjacent to a nucleosome-depleted region (NDR) at gene promoters. We demonstrate that SWR1 has a kinetic binding preference for DNA of NDR length as opposed to gene-body linker length DNA. Using single and dual color single-particle tracking on DNA stretched with optical tweezers, we directly observe SWR1 diffusion on DNA. We found that various factors impact SWR1 scanning, including ATP which promotes diffusion through nucleotide binding rather than ATP hydrolysis. A DNA-binding subunit, Swc2, plays an important role in the overall diffusive behavior of the complex, as the subunit in isolation retains similar, although faster, scanning properties as the whole remodeler. ATP-bound SWR1 slides until it encounters a protein roadblock, of which we tested dCas9 and nucleosomes. The median diffusion coefficient, 0.024 μm2/s, in the regime of helical sliding, would mediate rapid encounter of NDR-flanking nucleosomes at length scales found in cellular chromatin.

    1. Structural Biology and Molecular Biophysics
    Marina Schrecker et al.
    Research Article

    The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3’ end (3’ ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3’ ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.