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 electron paramagnetic resonance (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 toward 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.
Highlights
The passive permeation of low molecular weight solutes across the outer membrane (OM) of Gramnegative bacteria is typically facilitated by porins
Recent work has shown that the efficient incorporation of pairs of spin labels to make distance measurements using double electron-electron resonance (DEER) required the use of a strain deficient in the disulfide bond formation (Dsb) chaperone system (Nilaweera et al, 2019)
For sites 63, 72, and 93, the addition of vitamin B12 alters the spectra and increases the population of the immobile component indicating that incorporation of the label at these sites has not prevented the binding of substrate
Summary
The passive permeation of low molecular weight solutes across the outer membrane (OM) of Gramnegative bacteria is typically facilitated by porins. Many higher molecular weight solutes and trace nutrients, including carbohydrates, iron siderophores, cobalamin, copper, and nickel, are bound and transported across the OM by a family of active transporters that are TonB-dependent (Bolam and van den Berg, 2018; Noinaj et al, 2010). These TonB-dependent transporters (TBDTs) derive energy from the bacterial inner membrane by interacting with TonB, a transperiplasmic protein that interacts with the inner membrane proteins ExbB and ExbD (Celia et al, 2016; MakiYonekura et al, 2018). TonB interacts with the transporter through a conserved motif on the N-terminal side of the core termed the Ton box (Pawelek et al, 2006; Shultis et al, 2006)
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