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.
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.
- David S Cafiso
- David S Cafiso
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Janice L Robertson, Washington University in St Louis, United States
- Preprint posted: March 18, 2021 (view preprint)
- Received: March 18, 2021
- Accepted: July 11, 2021
- Accepted Manuscript published: July 12, 2021 (version 1)
- Accepted Manuscript updated: July 13, 2021 (version 2)
- Accepted Manuscript updated: July 30, 2021 (version 3)
- Version of Record published: August 5, 2021 (version 4)
© 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.
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SARS-CoV-2 spike protein plays a key role in mediating viral entry and inducing host immune responses. It can adopt either an open or closed conformation based on the position of its receptor-binding domain (RBD). It is yet unclear what cause these conformational changes or how they influence the spike's functions. Here we show that Lys417 in the RBD plays dual roles in the spike's structure: it stabilizes the closed conformation of the trimeric spike by mediating inter-spike-subunit interactions; it also directly interacts with ACE2 receptor. Hence, a K417V mutation has opposing effects on the spike's function: it opens up the spike for better ACE2 binding while weakening the RBD's direct binding to ACE2. The net outcomes of this mutation are to allow the spike to bind ACE2 with higher probability, mediate viral entry more efficiently, but become more exposed to neutralizing antibodies. Given that residue 417 has been a viral mutational hotspot, SARS-CoV-2 may have been evolving to strike a balance between infection potency and immune evasion, contributing to its pandemic spread.