Dynamic basis of lipopolysaccharide export by LptB₂FGC

Reviewer #1 (Public Review):

Summary:

The current manuscript uses electron spin resonance spectroscopy to understand how the dynamic behavior and conformational heterogeneity of the LPS transport system change during substrate transport and in response to the membrane, bound nucleotide (or transition state analog), and accessory subunits. The study builds on prior structural studies to expand our molecular understanding of this highly significant bacterial transport system.

Strengths

This series of well-designed and well-executed experiments provides new mechanistic insights into the dynamic behavior of the LPS transport system. Notable new insights provided by this study include its indication of the spatial organization of the LptC domain, which was poorly resolved in structures, and how the LptC domain modulates the dynamic behavior of the gate through which lipids access the binding site. In addition, a mass spectrometry approach designed to examine LPS binding at different stages in the nucleotide-dependent conformational cycle provides insight into the order of operations of LPS binding and transport.

Reviewer #2 (Public Review):

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and plays a critical role in bacterial virulence. The LPS export mechanism is a potential target for new antibiotics. Inhibiting this process can render bacteria more susceptible to the host immune system or other antibacterial agents. Given the rise of antibiotic-resistant bacteria, novel targets are urgently needed. The seven LPS transport (Lpt) proteins, A-G, move LPS from the inner to the outer membrane. This study investigated the conformational changes in the LptB2FG-LptC complex using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, revealing how ATP binding and hydrolysis affect the LptF β-jellyroll domain and lateral gates. The findings highlight the role of LptC in regulating LPS entry, ensuring efficient and unidirectional transport across the periplasm.

The β-jellyrolls are not fully resolved in the vanadate-trapped structure of LptB2FG and LptB2FGC. Therefore, the current study provides valuable information on the functional dynamics of these periplasmic domains, their interactions, and their roles in the unidirectional transport of LPS. Additionally, the dynamic perspective of the lateral gates in LptFG in the presence and absence of LptC is another strength of this study. Moreover, at least in detergent samples, more comprehensive intermediates of the ATP turnover cycle are studied than in the available structures, providing crucial missing mechanistic details.

Other major strengths of the study include high-quality DEER distance measurements in both detergent and proteoliposomes, the latter providing valuable dynamics information in the lipid environment. However, lipid composition is not mentioned. The proteoliposome study is crucial since the previous structural study (Li, Orlando & Liao 2019) was done in rather small-diameter nanodiscs, which might affect the overall dynamics of the complex. It would have been beneficial if the investigators had reconstituted the complex in lipid nanodiscs with the same composition as proteoliposomes. The mixed lipid/detergent micelles provide an alternative. It seems the ATPase activity of the protein complex is much lower in detergent compared with lipid nanodiscs (Li, Orlando & Liao 2019). In the current study, ATPase activity in proteoliposomes is not provided. Also, the reviewer assumes cysteine-less (CL) constructs of the complex components were utilized. The ATPase assay on CL complex is not presented.

Additionally, from previous structural studies and the mass spectrometry data presented here, LPS co-purifies and is already bound to the complex, thus the Apo state may represent the LPS-bound state without nucleotides.

The selection of sites to probe lateral gate 2, which forms the main LPS entry site, may pose an issue. Although the authors provide justification based on the available structures, one site (position 325 in LptF) is located on a flexible loop, and position 52 in LptG is on the neighboring transmembrane helix, separated by a potentially flexible loop from the gating TM1. These labeling sites could exhibit significant local dynamics, resulting in a broader distribution of distances and potentially masking the gating-related conformational changes.

Reviewer #3 (Public Review):

Summary:

The manuscript by Dajka and co-workers reports the application of a biophysical approach to analyse the dynamics of the LptB2FG-C ABC transporter, involved in LPS transport across the cell envelope in Escherichia coli. LptB2FG-C belongs to a new class of ABC transporters (type VI) and is essential and conserved in several Gram-negative pathogens. Since LPS is the major component of the outer membrane of the Gram-negative cell and is responsible for the low permeability of this membrane to several antibiotics, a deep understanding of the mechanism and function of the LptB2FG-C transporter is crucial for the development of new drugs targeting Gram-negative pathogens.

Several structural studies have been published so far on the LptB2FG-C transporter, disclosing important aspects of the transport mechanism; nevertheless, lack of resolution of some regions of the individual proteins as well as the dynamic nature of the transport mechanism per se (e.g. the insertion and removal of the TM helix of LptC from the TMDs of the transporter during the LPS transport cycle) has greatly limited the understanding of the mechanism that couples ATP binding and hydrolysis with LPS transport. This knowledge gap could be filled by applying an approach that allows the analysis of dynamic processes. The DEER/PELDOR technique applied in this work fits well with this requirement.

Strengths:

In this study, the authors provide some new pieces of information on the LptB2FG-C function and the role of LptC in the transporter. Notably, they show that:

-there is high heterogeneity in the conformational states of the entry gate of LPS in the transporter (gate-2) that are reduced by the insertion of LptC, and the heterogeneity observed is not altered by ATP binding or hydrolysis (as expected since LPS entry is ATP-independent).

-ATP binding induces an allosteric opening of LptF β-jellyroll domain that allows for LPS passage to the β-jellyroll of LptC, which is stably associated with the β-jellyroll of LptF throughout the cycle.

- the β-jellyroll of LptG is highly flexible, indicating an involvement in the LPS transport cycle.

The manuscript is timely and overall clear.

Weaknesses:

I list my concerns below and provide suggestions that, in my opinion, should be addressed to reinforce the findings of this study.

(1) Protein complex controls: the authors assess the ATPase activity of the spin-labelled variants of their protein complexes to rule out the possibility that engineering the proteins to enable spin labelling could affect their functionality (Figure S4). It has been reported that the association of LptC to LptB2FG complex inhibits its ATPase activity. However, in the ATPase assay data shown in Figure S4, the inhibitory effect of the LptC TM is not visible (please compare LptB2FG F-A45C G-I335C and F-L325C G-A52C with and without LptC). This can lead to suspect that the regulatory function of LptC is missing in the LptC-containing complexes used in this work. I suggest the authors include wt LptB2FGC in the assay to compare the ATPase activity of this complex with wt LptB2FG. The published inhibitory effect of TM LptC has been observed in proteoliposomes. Since it is not clear from the paper if the ATPase assay in Figure 4 has been conducted in DDM or proteoliposomes, the lack of inhibitory effect could be due to the assay conditions. A comparative test could answer this question.

(2) Figure 2: NBD closure upon ATP binding to LptB2FG is convincingly demonstrated both in DDM micelles and proteoliposomes, validating the experimental system. However, since under physiological conditions, ATP binding should take place before the displacement of the TM of LptC (Wilson and Ruiz, Mol microbiol 2022), I suggest the authors carry out the experiments with LptC-containing complexes to investigate conformational changes (if any) that are triggered when ATP binding occurs before the TM displacement.

(3) Proteoliposomes: in the experiments shown in Figures 3 and 4, unlike those in Figure 2, measurements in proteoliposomes give different results from the experiments in DDM, showing higher heterogeneity. Could this be related to the presence (or absence) of LPS in liposomes? It is not mentioned in the materials and methods section whether LPS is present. Could the authors please discuss this?

(4) The authors show large conformational heterogeneity in gate-2 (using the spin-labelled pair F-L325R1-G-A52R1) and suggest that deviation from the corresponding simulations could be due to the need for enhanced dynamics to allow for gate interaction with LPS or LptC. The effect of LptC is probed in the experiments shown in Figure 6, but I suggest the authors add LPS to the complexes to evaluate the possible stabilizing effect of LPS on the conformations shown in Figure 4.

(5) Figure 6: the measurement of lateral gate 1 and 2 dynamics in the LptC-containing complexes clearly supports the hypothesis, proposed based on the available structures, that TM LptC dissociates from LptB2FG upon ATP binding. However, direct evidence of this movement is still missing. Would it be possible to monitor the dynamics of the TM LptC by directly labelling this protein domain? This would give a conclusive demonstration of the displacement during the ATPase cycle.

(6) LPS release assay: Figure 6 panels H-I-J show the MS spectra relative to LPS-bound and free proteins obtained from wt LptB2FG upon ATP binding and ATP hydrolysis conditions. From these spectra the authors conclude that LPS is completely released only upon ATP hydrolysis. However, the current model predicts that LPS release into the Lpt bridge made by LptC-A-D is triggered by ATP binding. For this reason, I suggest the authors assess LPS release also from the LptB2FGC complex where, in the absence of LptA, LPS would be expected to be mostly retained by the complex under the same conditions.