Structures of LolB bound to LolA or lipoprotein resolve the final steps of bacterial lipoprotein trafficking

Reviewer #1 (Public review):

Summary:

The manuscript aims to mechanistically characterize the final step of the Lol lipoprotein trafficking pathway in Gram-negative bacteria, where triacylated lipoproteins are transferred from the periplasmic chaperone protein LolA to the outer membrane receptor LolB for subsequent insertion into the inner leaflet of the outer membrane. The crystal structures of LolA R43L-LolB, LolB L114G-lipoprotein, LolB Q44A (open), and LolB Q44A (closed) are reported. A combination of structural analysis, growth assays, pull-downs, and fluorescence spectroscopy was employed to characterize the behaviour of wild-type and mutant forms of Lol proteins. The authors then used previously published molecular dynamics simulations to propose a six-step model for the transfer and insertion of lipoproteins from LolA to LolB, and finally into the membrane.

The structural work is mostly solid, but the mechanistic conclusions drawn from it rely too much on interpolation between static states and on functional assays with limited dynamic range.

Strengths:

(1) The structure-guided mutagenesis was thorough and well designed. Mutants were tested both in isolation and in combination with one another, utilizing both alanine and charge reversal mutants, to fully tease apart which residues were most important for the interaction.

(2) The dissection of the Hook interactions allowed the authors to identify the importance of the identity of the hook apex residue, as well as the critical hydrogen bond that maintained the Hook geometry.

(3) The structure of the lipoprotein-bound LolB is a valuable addition to the body of knowledge around this pathway, and the way that the complex was produced was very clever.

Weaknesses:

(1) There is no raw data shown for any of the interactions or lipoprotein transfer experiments. All of the quantitative claims about LolA association and lipoprotein transfer rely on SDS-PAGE densitometry, and not a single gel image from those experiments is provided in the main text or supplemental.

(2) Pull-down assays are a fairly poor assay to use for protein interactions when there are so many quantitative alternatives that would be amenable to this experimental system, especially when the association and transfer of lipoprotein are the central claims of the manuscript.

(3) The crystal structures are under-refined for their resolution, with large R-factor gaps and somewhat concerning statistics contained in the wwPDB reports (e.g. >20% RSRZ outliers in 2/4 structures). The data also seem to be cut at strangely high I/sigI for the two Q44A structures with no explanation.

(4) In the LolB L114G structure, the Hook residue L68 is the most poorly fit in the whole structure. The validation report indicates the entire hook is poorly resolved, but without access to the maps and models, we are unable to assess this independently.

(5) There is no rubric for what constitutes a significant result for the DAUDA fluorescence spectroscopy. What level of increase vs wild-type is considered significant? I188G is incredibly close to L144G, but one is treated as a non-result and the other as something that should be further investigated.

(6) The conformational trajectory of the proposed model is on static endpoints.

Reviewer #2 (Public review):

Summary:

The manuscript by Jepson and colleagues presents the structural and biochemical study of the outer membrane LolB in complex with LolA in its apo and lipoprotein-bound states. The Lol system (LolABCDE) is essential in the translocation of lipoproteins to the outer membrane. Several studies have looked at the inner membrane complex. This study captured the last step of the process, the translocation of lipoproteins to the outer membrane via the LolB and LolA proteins. The crystal structures of LolB in complex with LolA revealed key interactions via the hook and charged belt motifs. Mutagenesis was not able to fully dissociate the complex or prevent the transfer of lipoprotein. The complex of LolB with LolA and lipoprotein revealed the 'final' step of engaging the complex for lipoprotein insertion into the outer membrane.

Overall, this is an excellent and exciting study for the Lol field as it captures the events happening at the outer membrane.

Strengths:

The authors have determined several high-resolution structures and validated them with excellent mutagenesis experiments. The key structure is that of LolB with LolA and the lipoprotein (even partially resolved).

Weaknesses:

The only 'weakness' is that the authors have used mutants of either LolB or LolA to capture the stable states, and the discussion section would benefit from commenting if these states could be dead-end states, as validation of the mutants has been done with a WT background. In addition, can they discuss whether a2 is likely to undergo further conformational changes to open a lateral gate to the OM for the acyl chains of the lipoprotein to insert?