Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that lipidated MetQ is required for interaction

  1. Naima G Sharaf
  2. Mona Shahgholi
  3. Esther Kim
  4. Jeffrey Y Lai
  5. David G VanderVelde
  6. Allen T Lee
  7. Douglas C Rees

  • Curated by eLife

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    Evaluation Summary:

    Sharaf and colleagues present an elegant structural and functional analysis of the Neisseria meningitidis ABC transporters MetQ/MetNI illustrating that the substrate binding protein MetQ requires N-terminal lipidation and a substrate (e.g. L-Met and other Met analogs) to stimulate the ATPase, presumably in order to transport the substrate across the inner membrane. This paper will be of broad interest to microbiologists and membrane physiologists who study periplasmic substrate binding proteins and transporter interactions in bacteria.

    (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 #3 agreed to share their name with the authors.)

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Abstract

NmMetQ is a substrate-binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the presence and absence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function and localization, playing a role in NmMetNI-mediated transport at the inner membrane and moonlighting on the bacterial surface.

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  1. Version published to 10.7554/elife.69742 on eLife
  2. eLife

    Author Response:

    Reviewer #2:

    To address the roles of NmMetQ protein, the authors used multiple biochemical and biophysical techniques to characterize the structure and function of NmMetQ without and with its cognate ABC transporter NmMetNI. However, considering the similar substrate binding protein EcMetQ from E. coli has been experimentally verified to be a lipoprotein, the major conclusion of this manuscript is not particularly novel. Besides, the authors should address some points to further strengthen their conclusion.

    Major points:

    1. The LC-MS results suggest that the recombinantly expressed and purified lipo-NmMetQ protein has lipid modifications, mainly deduced from the molecular masses. Did the authors perform other experiments to further support the presence of lipid modifications?

    In addition to using LC-MS to demonstrate that recombinantly expressed full-length NmMetQ leads to the production of lipid-modified NmMetQ, we changed the cysteine residue at position 20 to alanine. If NmMetQ was not a lipoprotein, we would expect the mass change to reflect the mass difference between cysteine and alanine. However, if full-length NmMetQ was a lipoprotein, this amino acid change would prevent lipid modification and lead to the accumulation of pre-protein NmMetQ. LC-MS analysis of both the full length and C20A NmMetQ proteins support our assertion that recombinant expression of full-length NmMetQ leads to the production of lipidated NmMetQ. Furthermore, the size exclusion chromatography trace illustrated in Figure 1B reveals that only a very small amount of unacylated wild-type protein is present (the small bump near 100 mL), indicating that the extent of lipidation of the wild-type protein is nearly complete.

    1. I noticed that the NmMetQC20A protein was also purified with DDM detergent, could the mutant protein be purified without detergent? And could the WT protein be purified without detergent? This experiment could be an additional evidence to support the absence of lipid modifications on the mutant protein and presence of lipid modifications on the WT protein.

    To maintain consistency between experiments, all proteins were purified in the presence of detergent. No attempts were made to purify NmMetQC20A or full length NmMetQ in the absence of detergent. We speculate that maximal extraction of lipo-NmMetQ and pre-protein NmMetQ would be difficult in the absence of detergent, since the lipid-moiety and the N-terminal signal sequence of these proteins are believed to be associated with membranes. However, secreted NmMetQ should readily purify in the absence of detergent, as we have observed with NmMetQ construct with the signal peptide truncated in our previous study (see reference below).

    Nguyen PT, Lai JY, Kaiser JT, Rees DC. Structures of the Neisseria meningitides methionine‐ binding protein MetQ in substrate‐ free form and bound to l‐ and d‐ methionine isomers. Protein Science. 2019 Oct;28(10):1750-7.

    1. It is very interesting to see that the lipid moiety of lipo-NmMetQ is required for maximal NmMetNI stimulation, especially compared to the secreted NmMetQ. This result suggests that the lipid moiety could participate in the NmMetNI stimulation directly. But the lipid moiety could not be resolved in the lipo-NmMetQ:NmMetNI complex structure, probably due to the limited resolution at 6.4 Å. This point is quite novel, unfortunately, this manuscript provided little insight on this.

    We agree that this is an important point; as noted above in our response to Essential Revision #1, it was not possible to increase the resolution of the lipo-NmMetQ:NmMetNI structure; in view of the observations by Liu et al also noted there, it is possible that the lipid is poorly ordered and not visible even in higher resolution structures.

    1. The inward-facing NmMetNI structure was resolved in the presence of lipo-NmMetQ and AMPPNP, but only the apo NmMetNI structure was captured. This is unexpected, and the authors should comment on this.

    Thank you for the suggestion. We have expanded our result section to comment on why we believe no complex formation was detected.

    1. The bioinformatic prediction of the distribution of lipid-modified MetQ proteins in different classes of Proteobacteria is very weak. As the authors mentioned in the Discussion part, future efforts should be made to experimentally determine which SBPs have lipid modifications. I would suggest that the authors should move the Fig. 5 to supplementary information and move the corresponding text to Discussion.

    We thank the reviewer for this suggestion. The observations from the bioinformatic analysis demonstrating that many Proteobacterial families can possess SBP lipoproteins, however, are important to establish the general relevance of our biochemical and structural findings. We therefore respectfully request that this data remain in the results section.

    Minor points:

    1. The authors should provide the details for cryo-EM sample preparations in the Methods and Materials, such as how the protein complex was assembled, the protein complex concentrations, AMP-PNP/ATP concentrations, methionine concentrations, et al.

    Thank you for noting this - we have expanded our sample preparation section to include the information requested.

    1. Please state the ATP concentration used in ATPase experiments in Methods and Materials.

    Thank you for noting this - we have added the information requested to the Methods and Materials.

  3. eLife

    Evaluation Summary:

    Sharaf and colleagues present an elegant structural and functional analysis of the Neisseria meningitidis ABC transporters MetQ/MetNI illustrating that the substrate binding protein MetQ requires N-terminal lipidation and a substrate (e.g. L-Met and other Met analogs) to stimulate the ATPase, presumably in order to transport the substrate across the inner membrane. This paper will be of broad interest to microbiologists and membrane physiologists who study periplasmic substrate binding proteins and transporter interactions in bacteria.

    (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 #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this study, Sharaf et al., characterize the role of the NmMetQ, a substrate binding protein (SBP) in Neisseria meningitidis, a gram-negative bacteria. Typically, SBPs are localized in the periplasmic space and play the primary role of sequestering rare substrates like amino acids, in this case methionine. Once bound, they bind to a receptor transporter, usually from the ATP binding cassette (ABC) transporter family, which hydrolyzes intracellular ATP to enable uptake of the substrate across the inner membrane. However, the SBP NmMetQ has raised some questions, since the protein has been found on the outer membrane of the cell and is a contender for a surface antigen for the development of meningococcal vaccines. To consolidate this finding, the researchers study NmMetQ to determine whether it still participates in modulating methionine transport. Through the use of the signal peptide server SignalP5.0, they hypothesize that NmMetQ is actually a lipoprotein, which they confirm by expression in E. coli and by mass spectrometry analysis. Then, to examine whether NmMetQ couples to the activity of the ABC transporter NmMetNI, they purify the transporter and measure ATPase activity in detergent micelles with and without NmMetQ. They find that ATP hydrolysis is strongly coupled to the presence of NmMetQ bound to methionine, indicating a preserved functional interaction between these proteins. Next, they investigate the binding of different methionine analogues to NmMetQ by "Fluorine chemical shift Anisotropy and eXchange for Screening (FAXS)", a rigorous screening method that shows a preference for the L-methionine form. Finally they determine cryoEM structures of the NmMetNI transporter at 3.6 Å resolution, and the NmMetQ complexed structure at > 6 Å. While the structural details are low for the complex, they are able to confirm binding as well as the global conformation. The studies clearly demonstrate that NmMetQ can act in the canonical mechanism as a sequestering protein that couples to activity of the ABC transporter. However, the discovery that these proteins are lipoproteins, and the analysis that other Proteobacterial families may possess similar lipoproteins, opens up a new area of biology, paving the way to a better understanding of why these SBPs are also surface localized. Altogether, the research that is presented is clear and rigorous, and substantially increases our understanding of SBP-transporter partnerships. This is a thoroughly interesting discovery.

  5. eLife

    Reviewer #2 (Public Review):

    To address the roles of NmMetQ protein, the authors used multiple biochemical and biophysical techniques to characterize the structure and function of NmMetQ without and with its cognate ABC transporter NmMetNI. However, considering the similar substrate binding protein EcMetQ from E. coli has been experimentally verified to be a lipoprotein, the major conclusion of this manuscript is not particularly novel. Besides, the authors should address some points to further strengthen their conclusion.

    Major points:

    1. The LC-MS results suggest that the recombinantly expressed and purified lipo-NmMetQ protein has lipid modifications, mainly deduced from the molecular masses. Did the authors perform other experiments to further support the presence of lipid modifications?

    2. I noticed that the NmMetQC20A protein was also purified with DDM detergent, could the mutant protein be purified without detergent? And could the WT protein be purified without detergent? This experiment could be an additional evidence to support the absence of lipid modifications on the mutant protein and presence of lipid modifications on the WT protein.

    3. It is very interesting to see that the lipid moiety of lipo-NmMetQ is required for maximal NmMetNI stimulation, especially compared to the secreted NmMetQ. This result suggests that the lipid moiety could participate in the NmMetNI stimulation directly. But the lipid moiety could not be resolved in the lipo-NmMetQ:NmMetNI complex structure, probably due to the limited resolution at 6.4 Å. This point is quite novel, unfortunately, this manuscript provided little insight on this.

    4. The inward-facing NmMetNI structure was resolved in the presence of lipo-NmMetQ and AMPPNP, but only the apo NmMetNI structure was captured. This is unexpected, and the authors should comment on this.

    5. The bioinformatic prediction of the distribution of lipid-modified MetQ proteins in different classes of Proteobacteria is very weak. As the authors mentioned in the Discussion part, future efforts should be made to experimentally determine which SBPs have lipid modifications. I would suggest that the authors should move the Fig. 5 to supplementary information and move the corresponding text to Discussion.

    Minor points:

    1. The authors should provide the details for cryo-EM sample preparations in the Methods and Materials, such as how the protein complex was assembled, the protein complex concentrations, AMP-PNP/ATP concentrations, methionine concentrations, et al.

    2. Please state the ATP concentration used in ATPase experiments in Methods and Materials.

  6. eLife

    Reviewer #3 (Public Review):

    Although the lipidated MetQ protein is isolated from E.coli and not the native Neisseria, this reviewer is satisfied that the MetQ proteins lipidation state is valid when produced in E.coli.

    The ATPase assay of MetNI coupled to lipidated MetQ (MET) binding is a nice way of testing binding and correlating this with the F-NMR FAXS data to examine substrate specificity and diversity is clever.

    The authors present 2 Cryo-EM structures of NmMetNI in the inward-facing and outward facing (with MetQ bound) forms. The inward facing adds to our understanding of how interactions between the NBDs of ABC transporters without a C2 autoinhibitory domain differ from their homologous structures with autoinhibitory domains.

    Enthusiasm for this study is slightly dampened by the lack of resolution of the ternary complex that doesn't give insights on how lipidated MetQ binds to MetNI whereas the nonlipidated form does not stimulate activity. This would have been a nice structural outcome. The lower resolution structure of the Lipidated MetQ bound to MetNI would have been more impactful if we could see how/why the lipid on MetQ is required for stimulation of ATPase activity or if it told us whether NmMETQ is located in the inner leaflet of the OM or outer leaflet of the IM - presumable it is the latter. But this highlights an important question: what regulates the transport of lipidated-MetQ to the OM -is it the presence of substrate Met and its interaction with the integral membrane protein MetNI that retains it?

    Given all this work, the title could be changed as it should reflect that the characterization of the ABC methionine transporter from Neisseria reveals that Lipidated MetQ is required for interaction.

    Overall this is an important contribution to our understanding of the function of lipidated MetQ as a SBP and component of an ABC importer in Neisseria. Especially as MetQ is being considered as a vaccine candidate to prevent Neisseria infections and groups are working towards this goal.

  7. Version published to 10.1101/2021.05.04.442564v1 on bioRxiv