PREreview of Structural basis of lipopolysaccharide translocon assembly mediated by the small lipoprotein LptM

In this manuscript, Miyazaki et al. studied the interactions between EcLptM and EcLptD/E, by performing mutagenesis experiments, immunoblotting, crosslinking assays and solving the structure of the EcLptD/E/M complex via cryoEM, to further understand the role of LptM in LptD assembly and maturation. This study revealed that LptM has an essential region (C₂₀GLKGPLYF₂₈) within its N-terminal domain that interacts with LptD, although possibly slightly longer as discussed below. Consistent with the resolved cryoEM structure of EcLptD/E/M, in vivo disulfide crosslinking experiments revealed that LptM residue F28 is involved in the interaction with the LptD barrel domain. Additionally, residue G21 was shown to be critical for the function of LptM in the maturation of LptD. Also, the authors revealed the timing at which LptM interacts with LptD, showing that it acts at the late maturation step of LptD, after the action of BepA. The manuscript is well-written, and the conclusions made are supported by the data presented. We provide major and minor comments to help clarify some of the data and interpretations made.

Major comments

Figure 1C. Could you explain why erythromycin sensitivity increased in the ΔbepA strain, while it resembled that of WT in the ΔlptM strain? Does it mean that maintaining a well-folded β-barrel domain is sufficient to maintain OM integrity? This is beyond the scope of this study, but do you think that would also be the case inΔdsbA strain?

Figure 1E-F. Narita et al., 2016 concluded that BepA might play a role in facilitating the interaction of LptD and LptE at the BAM complex. This conclusion was based on the observed suppression of erythromycin sensitivity when overexpressing lptE in a ΔbepA strain which you also observed. Consider including this hypothesis as part of the discussion.

Figure 2B. Could you explain the accumulation of LptD^C in K33amb and A35amb strains? Could it mean that LptM becomes less stable, or are these residues essential for LptM function?

Lines 174-183 and lines 248-250: The interpretation that LptM in the cryoEM model stabilizes the folding of LptD due to the observed tight closure of the β-barrel junction is not fully convincing. It was based on comparisons made with the crystal structure of S. flexneri and an Alpha-fold model. Would it be possible to perform the comparison with a cryoEM structure of EcLptD/E without LptM instead, to see whether the junction is tightly closed in the absence of LptM or not? Or maybe do the comparison with the solved crystal structure (only β-barrel domain) of E. coli (PDB ID: 4RHB)?

Figure 4B. Consider showing the +ME lane between 15 and 20 KDa with αFLAG antibody to control that LptM is present.

Minor comments

Introduction (Lines 80-88): Could you add to the LptM paragraph that it interacts with the LptD/E translocon by mimicking LPS binding?

Figure S4A (middle). The crystal structure presented (PDB ID: 4q35) is for Shigella flexneri and not E. coli.

Lines 176-177: The crystal structure solved in Qiao et al., 2014 is for Shigella flexneri and not E. coli.

Figure 5 and Lines 208-209: Could you clarify why alanine, cysteine and tryptophan were chosen for the mutagenesis experiment? Is it because they represent a non-polar (small), polar and non-polar (bulky) amino acids, respectively? Also, the data shows that only when G21 was mutated to Trp (and not to Ala or Cys), was the LptM activity affected. Could you explain why? And whether it has to do with steric hinderance or that the bulkiness of Trp obscures an essential interaction between other two amino acid residues? Could you show with what residues of the β-barrel domain G21 interacts in the cryoEM structure?

Also, could you show an alignment of the conserved region with LptM homologs in the Enterobacteriaceae family and show whether G21 is conserved?

Lines 243-245: “Considering that this short essential region tightly interacts with the β-barrel domain of LptD (Figure 3, 4), it is unlikely to serve as a recruiter for the disulfide oxidase DsbA or disulfide isomerase DsbC to the LptD intermediate”, Could you explain why being in tight interaction with the β-barrel domain rules out the possibility that LptM recruits either DsbA or DsbC? Also, could you re-type “Considering that this short essential region....” to “considering the short essential region of LptM ....”, because it is not clear to the reader if you were referring to LptM.

Section 5 (Lines 218-250): Since this section describes the model, could you make the model a main figure instead of a supporting figure?

Lines 261-263: Could you remove “consisting almost entirely of signal sequences”, because, for example, LptM is not made entirely from a signal sequence or clarify that you are referring to the secreted proteins.

Sally Abulaila, Kim Kissoon and Habib Ogunyemi (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from group discussion including Michael Kwakye, Madaline McPherson, Madison McReynolds, Mandkhai Molomjamts, Octavio Origel and Warren Wilson.

Competing interests

The authors declare that they have no competing interests.