1. Microbiology and Infectious Disease
  2. Structural Biology and Molecular Biophysics
Download icon

Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport

  1. Thushani D Nilaweera
  2. David A Nyenhuis
  3. David S Cafiso  Is a corresponding author
  1. University of Virginia, United States
Research Article
  • Cited 0
  • Views 272
  • Annotations
Cite this article as: eLife 2021;10:e68548 doi: 10.7554/eLife.68548

Abstract

Outer membrane TonB-dependent transporters facilitate the uptake of trace nutrients and carbohydrates in Gram negative bacteria and are essential for pathogenic bacteria and the health of the microbiome. Despite this, their mechanism of transport is still unknown. Here, pulse EPR measurements were made in intact cells on the Escherichia coli vitamin B12 transporter, BtuB. Substrate binding was found to alter the C-terminal region of the core and shift an extracellular substrate binding loop 2 nm towards the periplasm; moreover, this structural transition is regulated by an ionic lock that is broken upon binding of the inner membrane protein TonB. Significantly, this structural transition is not observed when BtuB is reconstituted into phospholipid bilayers. These measurements suggest an alternative to existing models of transport, and they demonstrate the importance of studying outer membrane proteins in their native environment.

Data availability

Raw unprocessed DEER data are available in a compressed folder called "SourceData". The Pymol session file used to produce Fig. 6b is included as a supplementary file.

Article and author information

Author details

  1. Thushani D Nilaweera

    Genetics and Biochemistry Branch, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. David A Nyenhuis

    Department of Chemistry and Center for Membrane Biology, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. David S Cafiso

    Chemistry, University of Virginia, Charlottesville, United States
    For correspondence
    dsc0b@virginia.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3813-8721

Funding

Office of Extramural Research, National Institutes of Health (NIGMS GM035215)

  • David S Cafiso

Office of Extramural Research, National Institutes of Health (NIGMS S10OD025149)

  • David S Cafiso

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

Reviewing Editor

  1. Janice L Robertson, Washington University in St Louis, United States

Publication history

  1. Received: March 18, 2021
  2. Accepted: July 11, 2021
  3. Accepted Manuscript published: July 12, 2021 (version 1)
  4. Accepted Manuscript updated: July 13, 2021 (version 2)
  5. Accepted Manuscript updated: July 30, 2021 (version 3)

Copyright

© 2021, Nilaweera 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

  • 272
    Page views
  • 65
    Downloads
  • 0
    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. Evolutionary Biology
    2. Microbiology and Infectious Disease
    Alvin X Han et al.
    Research Article

    The evolution of influenza viruses is fundamentally shaped by within-host processes. However, the within-host evolutionary dynamics of influenza viruses remain incompletely understood, in part because most studies have focused on infections in healthy adults based on single timepoint data. Here, we analysed the within-host evolution of 82 longitudinally-sampled individuals, mostly young children, infected with A/H1N1pdm09 or A/H3N2 viruses between 2007 and 2009. For A/H1N1pdm09 infections during the 2009 pandemic, nonsynonymous minority variants were more prevalent than synonymous ones. For A/H3N2 viruses in young children, early infection was dominated by purifying selection. As these infections progressed, nonsynonymous variants typically increased in frequency even when within-host virus titres decreased. Unlike the short-lived infections of adults where de novo within-host variants are rare, longer infections in young children allow for the maintenance of virus diversity via mutation-selection balance creating potentially important opportunities for within-host virus evolution.

    1. Computational and Systems Biology
    2. Microbiology and Infectious Disease
    Wellington Miranda S et al.
    Research Article Updated

    Many bacteria communicate with kin and coordinate group behaviors through a form of cell-cell signaling called acyl-homoserine lactone (AHL) quorum sensing (QS). In these systems, a signal synthase produces an AHL to which its paired receptor selectively responds. Selectivity is fundamental to cell signaling. Despite its importance, it has been challenging to determine how this selectivity is achieved and how AHL QS systems evolve and diversify. We hypothesized that we could use covariation within the protein sequences of AHL synthases and receptors to identify selectivity residues. We began by identifying about 6000 unique synthase-receptor pairs. We then used the protein sequences of these pairs to identify covariation patterns and mapped the patterns onto the LasI/R system from Pseudomonas aeruginosa PAO1. The covarying residues in both proteins cluster around the ligand-binding sites. We demonstrate that these residues are involved in system selectivity toward the cognate signal and go on to engineer the Las system to both produce and respond to an alternate AHL signal. We have thus demonstrated that covariation methods provide a powerful approach for investigating selectivity in protein-small molecule interactions and have deepened our understanding of how communication systems evolve and diversify.