Nyenhuis, D. A. | bioRxiv Science

bioRxiv2021

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

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, where TonB binding alone is sufficient to produce allosteric rearrangements in the transporter. They also demonstrate the importance of studying outer membrane proteins in their native environment.

biophysics↗

bioRxiv2019

Disulfide chaperone knock-outs enable in-vivo double spin-labeling of an outer-membrane transporter

Recent advances in the application of EPR spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer-membrane proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli outer-membrane vitamin B12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated outer membranes or reconstituted systems, and the ability to label and perform EPR in cells is expected to be applicable to a range of other bacterial outer-membrane proteins.\n\nStatement of SignificanceEPR spectroscopy is an important method to characterize the structure and dynamics of membrane proteins, and recent efforts demonstrate that pulse EPR can be used to examine the extracellular surface of outer membrane proteins in live bacteria. In the present work, we show that pairs of cysteine residues in the Escherichia coli vitamin B12 transporter, BtuB, cannot be spin-labeled in wild-type strains, but can be labeled with the use of certain null mutants in the periplasmic disulfide bond formation, Dsb, system. These mutants also facilitate efficient spin-labeling of cysteines located on the periplasmic surface of BtuB. Distance measurements using pulse EPR provide evidence that the behavior of BtuB is different in the bacterial cell than it is in purified systems.

biophysics↗