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

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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.

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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.

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Thushani D Nilaweera

Genetics and Biochemistry Branch, University of Virginia, Charlottesville, United States

Competing interests
The authors declare that no competing interests exist.
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.
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.

"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.

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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.