Simultaneous Recognition of Multiple Signals in Bacterial Outer Membrane Proteins Enhance Assembly and Maintain Membrane Integrity

Reviewer #1 (Public Review):

Summary:
The biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria is still not fully understood, particularly substrate recognition and insertion by beta-assembly machinery (BAM). In the studies, the authors present their studies that in addition to recognition by the last strand of an OMP, sometimes referred to as the beta-signal, an additional signal upstream of the last strand is also important for OMP biogenesis.

Strengths:
1. Overall the manuscript is well organized and written, and addresses an important question in the field. The idea that BAM recognizes multiple signals on OMPs has been presented previously, however, it was not fully tested.

2. The authors here re-address this idea and propose that it is a more general mechanism used by BAM for OMP biogenesis.

3. The notion that additional signals assist in biogenesis is an important concept that indeed needs fully tested in OMP biogenesis.

4. A significant study was performed with extensive experiments reported in an attempt to address this important question in the field.

5. The identification of important crosslinks and regions of substrates and Bam proteins that interact during biogenesis is an important contribution that gives clues to the path substrates take en route to the membrane.

Weaknesses:

Major critiques (in no particular order):

1. The title indicates 'simultaneous recognition', however no experiments were presented that test the order of interactions during OMP biogenesis.

2. Aspects of the study focus on the peptides that appear to inhibit OmpC assembly, but should also include an analysis of the peptides that do not to determine this the motif(s) present still or not.

3. The b-signal is known to form a b-strand, therefore it is unclear why the authors did not choose to chop OmpC up according to its strands, rather than by a fixed peptide size. What was the rationale for how the peptide lengths were chosen since many of them partially overlap known strands, and only partially (2 residues) overlap each other? It may not be too surprising that most of the inhibitory peptides consist of full strands (#4, 10, 21, 23).

4. It would be good to have an idea of the propensity of the chosen peptides to form b-stands and participate in b-augmentation. We know from previous studies with darobactin and other peptides that they can inhibit OMP assembly by competing with substrates.

5. The recognition motifs that the authors present span up to 9 residues which would suggest a relatively large binding surface, however, the structures of these regions are not large enough to accommodate these large peptides.

6. The authors highlight that the sequence motifs are common among the inhibiting peptides, but do not test if this is a necessary motif to mediate the interactions. It would have been good to see if a library of non-OMP related peptides that match this motif could also inhibit or not.

7. In the studies that disrupt the motifs by mutagenesis, an effect was observed and attributed to disruption of the interaction of the 'internal signal'. However, the literature is filled with point mutations in OMPs that disrupt biogenesis, particular those within the membrane region. F280, Y286, V359, and Y365 are all residues that are in the membrane region that point into the membrane. Therefore, more work is needed to confirm that these mutations are in parts of a recognition motif rather than on the residues that are disrupting stability/assembly into the membrane.

8. The title of Figure 3 indicates that disrupting the internal signal motif disrupts OMP assembly, however, the point mutations did not seem to have any effect. Only when both 280 and 286 were mutated was an effect observed. And even then, the trimer appeared to form just fine, albeit at reduced levels, indicating assembly is just fine, rather the rate of biogenesis is being affected.

9. In Figure 4, the authors attempt to quantify their blots. However, this seems to be a difficult task given the lack of quality of the blots and the spread of the intended signals, particularly of the 'int' bands. However, the more disturbing trend is the obvious reduction in signal from the post-urea treatment, even for the WT samples. The authors are using urea washes to indicate removal of only stalled substrates. However a reduction of signal is also observed for the WT. The authors should quantify this blot as well, but it is clear visually that both WT and the mutant have obvious reductions in the observable signals. Further, this data seems to conflict with Fig 3D where no noticeable difference in OmpC assembly was observed between WT and Y286A, why is this the case?

10. The pull down assays with BamA and BamD should include a no protein control at the least to confirm there is no non-specific binding to the resin. Also, no detergent was mentioned as part of the pull downs that contained BamA or OmpC, nor was it detailed if OmpC was urea solubilized.

11. The neutron reflectometry experiments are not convincing primarily due to the lack controls to confirm a consistent uniform bilayer is being formed and even if so, uniform orientations of the BamA molecules across the surface. Further, no controls were performed with BamD alone, or with OmpC alone, and it is hard to understand how the method can discriminate between an actual BamA/BamD complex versus BamA and BamD individually being located at the membrane surface without forming an actual complex. Previous studies have reported difficulty in preparing a complex with BamA and BamD from purified components. Additionally, little signal differences were observed for the addition of OmpC. However, an elongated unfolded polypeptide that is nearly 400 residues long would be expected to produce a large distinct signal given that only the C-terminal portion is supposedly anchored to BAM, while the rest would be extended out above the surface. The depiction in Figure 5D is quite misleading when viewing the full structures on the same scales with one another.

12. In the crosslinking studies, the authors show 17 crosslinking sites (43% of all tested) on BamD crosslinked with OmpC. Given that the authors are presenting specific interactions between the two proteins, this is worrisome as the crosslinks were found across the entire surface of BamD. How do the authors explain this? Are all these specific or non-specific?

13. The study in Figure 6 focuses on defined regions within the OmpC sequence, but a more broad range is necessary to demonstrate specificity to these regions vs binding to other regions of the sequence as well. If the authors wish to demonstrate a specific interaction to this motif, they need to show no binding to other regions.

14. The levels of the crosslinks are barely detectable via western blot analysis. If the interactions between the two surfaces are required, why are the levels for most of the blots so low?

15. Figure 7 indicates that two regions of BamD promote OMP orientation and assembly, however, none of the experiments appears to measure OMP orientation? Also, one common observation from panel F was that not only was the trimer reduced, but also the monomer. But even then, still a percentage of the trimer is formed, not a complete loss.

16. The experiment in Fig 7B would be more conclusive if it was repeated with both the Y62A and R197A mutants and a double mutant. These controls would also help resolve any effect from crowding that may also promote the crosslinks. Further, the mutation of R197 is an odd choice given that this residue has been studied previously and was found to mediate a salt bridge with BamA. How was this resolved by the authors in choosing this site since it was not one of the original crosslinking sites?

17. As demonstrated by the authors in Fig 8, the mutations in BamD lead to reduction in OMP levels for more than just OmpC and issues with the membrane are clearly observable with Y62A, although not with R197A in the presence of VCN. The authors should also test with rifampicin which is smaller and would monitor even more subtle issues with the membrane. Oddly, no growth was observed for the Vec control in the lower concentration of VCN, but was near WT levels for 3 times VCN, how is this explained?

18. While Fig 8I indeed shows diminished levels for FY as stated, little difference was observed for the trimer for the other mutants compared to WT, although differences were observed for the dimer. Interestingly, the VY mutant has nearly WT levels of dimer. What do the authors postulate is going on here with the dimer to trimer transition? How do the levels of monomer compare, which is not shown?

19. In the discussion, the authors indicate they have '...defined an internal signal for OMP assembly', however, their study is limited and only investigates a specific region of OmpC. More is needed to definitively say this for even OmpC, and even more so to indicate this is a general feature for all OMPs.

20. In the proposed model in Fig 9, it is hard to conceive how 5 strands will form along BamD given the limited surface area and tight space beneath BAM. More concerning is that the two proposal interaction sites on BamD, Y62 and R197, are on opposite sides of the BamD structure, not along the same interface, which makes this model even more unlikely. As evidence against this model, in Figure 9E, the two indicates sites of BamD are not even in close proximity of the modeled substrate strands.

Reviewer #2 (Public Review):

Previously, using bioinformatics study, authors have identified potential sequence motifs that are common to a large subset of beta-barrel outer membrane proteins in gram negative bacteria. Interestingly, in that study, some of those motifs are located in the internal strands of barrels (not near the termini), in addition to the well-known "beta-signal" motif in the C-terminal region.

Here, the authors carried out rigorous biochemical, biophysical, and genetic studies to prove that the newly identified internal motifs are critical to the assembly of outer membrane proteins and the interaction with the BAM complex. The author's approaches are rigorous and comprehensive, whose results reasonably well support the conclusions. While overall enthusiastic, I have some scientific concerns with the rationale of the neutron refractory study, and the distinction between "the intrinsic impairment of the barrel" vs "the impairment of interaction with BAM" that the internal signal may play a role in. I hope that the authors will be able to address this.

Strengths:

1. It is impressive that the authors took multi-faceted approaches using the assays on reconstituted, cell-based, and population-level (growth) systems.

2. Assessing the role of the internal motifs in the assembly of model OMPs in the absence and presence of BAM machinery was a nice approach for a precise definition of the role.

Weaknesses:

1. The result section employing the neutron refractory (NR) needs to be clarified and strengthened in the main text (from line 226). In the current form, the NR result seems not so convincing.

What is the rationale of the approach using NR?
What is the molecular event (readout) that the method detects?
What are "R"-y axis and "Q"-x axis and their physical meanings (Fig. 5b)?
How are the "layers" defined from the plot (Fig. 5b)?
What are the meanings of "thickness" and "roughness" (Fig. 5c)?
What are the meanings of the increases in thickness and roughness?
What does "SLD" stand for?

2. In the result section, "The internal signal is necessary for insertion step of assembly into OM"

This section presents an important result that the internal beta-signal is critical to the intrinsic propensity of barrel formation, distinct from the recognition by BAM complex. However, this point is not elaborated in this section. For example, what is the role of these critical residues in the barrel structure formation? That is, are they involved in any special tertiary contacts in the structure or in membrane anchoring of the nascent polypeptide chains?