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

  • Curated by eLife

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    Evaluation Summary:

    The manuscript endeavors to explain the mechanism of action of the gram-negative bacterial outer membrane TonB-dependent transporter BtuB, which acquires vitamin B12 from the external environment. The authors use electron paramagnetic resonance spectroscopy to monitor the proximity of different parts of this protein to one another during the binding of B12 directly in the E. coli outer membrane. This manuscript will be of interest to those who study the biophysics of membrane transporters and stresses the importance of studying membrane proteins in their native environment.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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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 electron paramagnetic resonance (EPR) measurements were made in intact cells on the Escherichia coli vitamin B 12 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.

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  1. eLife

    Author Response

    Reviewer # 1 stated that “…some of the conclusions are premature, especially concerning the mechanism of action of the Ton complex in the catalyzed transport of vitB12. While the data show clear differences between the apo and vitB12 bound states of BtuB, the conclusions on the actual transport mechanism of vitB12 into the periplasm are more speculative.”

    We agree that these conclusions regarding the mechanism of action of TonB are more speculative, and we eliminated this part of the discussion and re-written two paragraphs towards the end of the discussion.

    The main concern of this reviewer is the conclusions reached from the data obtained with the R14A and/or D316A mutants. There is a clear dramatic change of conformation for the SB3 loop for these mutants upon substrate binding, and as shown the natural environment of BtuB is important to detect these changes. However, the authors state that "breaking the ionic lock and eliminate the electrostatic interactions of R14, as we have done here, should mimic the TonB bound state" (lines 487-488). The data presented in the manuscript do not support this statement as they do not allow to monitor the structural state of the N-terminal TonB box.

    The reviewer is correct that we do not know the precise state of the Ton box in this in-vivo system. All we can say is that one outcome of TonB binding would be to eliminate the electrostatic interactions that we have eliminated or reduced by mutation. In the present experiment, BtuB is in excess (by maybe a factor of 20 to 50) over TonB; as a result, populating a TonB bound state of BtuB to levels sufficient for spectroscopic measurements may not be possible (without modifying the system). We have removed the statement and re-written the paragraph in the discussion.

    Later it is proposed that "the movement of SB3 may also drive the movement of substrate" (lines 466-467) and that "this structural change may be sufficient to move the substrate into the periplasm" (lines 501-502). This is highly speculative, as in the structural states observed with the broken ionic lock, it cannot be determined if vitB12 is still bound to BtuB, or released in the periplasm. As noted, the "conversion of the transporter to the apo state does not occur under the conditions of the experiment" (lines 398-399). It is possible that this structural state is locked with a vitB12 bound and unable to complete the transport cycle.

    The reviewer is correct to state that B12 may be in a locked state. However, the fact that the transporter does not appear to cycle back to its apo state may have more to do with the high ratio of BtuB to TonB and the slowness of the transport system. But the reviewers’ point is valid and until we know more about how these mutants alter transport and can perform experiments to track the position of the substrate, the statement is speculative. We have removed it from the discussion.

  2. Version published to 10.7554/elife.68548 on eLife
  3. eLife

    Reviewer #3 (Public Review):

    The manuscript endeavors to explain the mechanism of action of a Gram-negative bacterial outer membrane (OM) TonB-dependent transporter (TBDT), that acquires metabolites (in this case vitamin B12) from the external environment. The authors use electron paramagnetic resonance spectroscopy to monitor the proximity of different parts of OM protein to one another during the binding of B12. Their data show that different conformations of the target protein occur during the binding of B12.

  4. eLife

    Reviewer #2 (Public Review):

    This study aims at elucidating the substrate-dependent conformational dynamics of TonB-dependent transporter BtuB, which is responsible for vitamin B12 transport in the outer membrane of E. coli. Following the pioneering studies from the same lab, the study employs an innovative approach of in-situ site-directed spin labeling for CW EPR spectroscopy and double electron-electron resonance (DEER) distance measurements in intact E. coli. Despite the intricacy of spin labeling and performing DEER measurements in intact cells, the majority of the obtained DEER spectra are of high quality with impressively long dipolar evolution times and signal-to-noise ratio, enhancing the reliability and accuracy of the data. Despite the limited number of distance constraints on one side of the core domain, experiments are well designed to address the relevant questions and the conclusions are justified by the data. The results fully support the main conclusion that the large substrate-induced structural change on the C-terminal side of the core in the presence of the mutations that mimic the breakage of the R14-D316 ionic lock (i.e., R14A, D316A, D316A/R14A), indicates the shift of the substrate-binding loop 3 towards the periplasm and reproduces the state when the transporter is bound to both substrate and TonB. The authors have utilized the deduced information to assess the currently proposed transport mechanisms. This study provides evidence for a transport mechanism that does not require a mechanical pulling or rotation by TonB as previously proposed. This model is also compatible with the structure of BtuB in complex with TonB that indicates the TonB-dependent release of the ionic lock. It is notable that these results are not seen in reconstituted membranes further highlighting the significance of in-situ structural dynamics studies in general and specifically for the field of EPR spectroscopy. I see substantial advance with respect to, both the mechanism of membrane transport by the TonB-dependent transporters and the application of this innovative approach.

  5. eLife

    Reviewer #1 (Public Review):

    This paper investigates the structural conformation of BtuB, a membrane protein involved in the intracellular transport of vitamin B12 in bacteria, using DEER techniques through the labelling of targeted pairs of amino acids. Using these techniques on whole cells, they detect structural conformations of the transporter upon binding of its substrate vitB12 that were not detected when BtuB is not in a natural environment.

    BtuB belongs to a well-known family of transporters found in the outer membrane of Gram-negative bacteria, called TBDTs (TonB Dependent Transporters). The structure of BtuB has been determined by X-ray crystallography in its apo and vitB12 bound states, as well as interacting with a C-terminal domain of the TonB protein, a periplasmic protein linked to the inner membrane that conveys the energy needed for the transport of vitB12 through BtuB. The TBDTs share a conserved architecture comprising a C-terminal 22 ß-barrel ring occluded by a globular N-terminal domain.

    Using EPR techniques, the Cafiso's lab has shown previously that the BtuB structural states are somewhat different or more dynamic than the X-ray structures would suggest. In the present paper they show that the Substrate Binding SB3 loop of BtuB, upon vitB12 binding, adopts some structural conformations in whole cells that are different than observed with BtuB reconstituted in liposomes, showing that a non-natural environment might alter its functionality. By using a set of mutants that supposedly mimics the BtuB-TonB state, they find dramatic conformational changes of the SB3 loop, which might represent an intermediate conformation of the open state of BtuB, in which vitB12 is allowed to move toward the periplasm, bypassing the need of an energized Ton complex. The data presented are convincing and show that the natural environment of BtuB, i.e., an intact outer membrane, but also probably some periplasmic components such as peptidoglycan and the Ton complex, influence the structural state of this protein. Most importantly these states are not detected when BtuB is reconstituted into liposomes, stressing the importance of studying these transporters in whole cells. However, some of the conclusions are premature, especially concerning the mechanism of action of the Ton complex in the catalyzed transport of vitB12. While the data show clear differences between the apo and vitB12 bound states of BtuB, the conclusions on the actual transport mechanism of vitB12 into the periplasm are more speculative.

    The main concern of this reviewer is the conclusions reached from the data obtained with the R14A and/or D316A mutants. There is a clear dramatic change of conformation for the SB3 loop for these mutants upon substrate binding, and as shown the natural environment of BtuB is important to detect these changes. However, the authors state that "breaking the ionic lock and eliminate the electrostatic interactions of R14, as we have done here, should mimic the TonB bound state" (lines 487-488). The data presented in the manuscript do not support this statement as they do not allow to monitor the structural state of the N-terminal TonB box.

    Later it is proposed that "the movement of SB3 may also drive the movement of substrate" (lines 466-467) and that "this structural change may be sufficient to move the substrate into the periplasm" (lines 501-502). This is highly speculative, as in the structural states observed with the broken ionic lock, it cannot be determined if vitB12 is still bound to BtuB, or released in the periplasm. As noted, the "conversion of the transporter to the apo state does not occur under the conditions of the experiment" (lines 398-399). It is possible that this structural state is locked with a vitB12 bound and unable to complete the transport cycle.

    Nevertheless, these mutants affecting the ionic lock seem to represent valuable tools to investigate the structural intermediates during transport. It remains to be seen if these mutants still promote transport in vivo.

  6. eLife

    Evaluation Summary:

    The manuscript endeavors to explain the mechanism of action of the gram-negative bacterial outer membrane TonB-dependent transporter BtuB, which acquires vitamin B12 from the external environment. The authors use electron paramagnetic resonance spectroscopy to monitor the proximity of different parts of this protein to one another during the binding of B12 directly in the E. coli outer membrane. This manuscript will be of interest to those who study the biophysics of membrane transporters and stresses the importance of studying membrane proteins in their native environment.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

  7. Version published to 10.1101/2021.03.18.436049v1 on bioRxiv