Lipoprotein trafficking in E. coli via the Lol pathway.

1) LolA is recruited by the LolCDE ABC transporter through interaction with the ‘Hook’ and ‘Pad’ structural features of LolC (27). Lipoprotein (LP) acyl chains bind at the interface of LolC and LolE (28). Binding of ATP to the nucleotide-binding protein LolD and subsequent nucleotide hydrolysis triggers lipoprotein transfer to LolA and resets the LolCDE transporter for a new cycle. 2) The lipoprotein acyl chains are enclosed inside the LolA cavity (32) forming a soluble complex able to cross the periplasm. 3) The lipoprotein-LolA complex interacts with outer membrane anchored-LolB, and lipoprotein transfer occurs between the LolA and LolB proteins. 4) The hydrophobic cavity of LolB is hypothesized to accommodate the lipoprotein acyl chains prior to insertion into the inner leaflet of the outer membrane.

Crystal structure of LolB bound to LolA R43L.

(A) Overall views of the complex with LolA R43L in blue and LolB in yellow. The positions of LolB ‘Hook’ and ‘Belt’ are indicated. Main chains of LolB Belt residues are shown as black transparent surface. (B) Surface representation of the LolA R43L-LolB complex. Dashed lines indicate the axis of each cavity, highlighting the 105° angle between the two protein barrels. A view of the LolA-LolB cavity entrance is shown in inset. (C) Close-up views of the boxed regions in A showing the interactions between LolA and LolB.

Role of the LolB Hook and Belt in the LolA-LolB interaction.

(A) In vitro interaction and lipoprotein transfer (dark and light grey respectively) between LolA and wild-type LolB (WT) or indicated variants. His-tagged LolB proteins were incubated with tag-free LolA or LolA-Pal complex prior to immobilization on Ni resin. After washing, bound proteins were eluted and quantified by SDS-PAGE. Data were normalized against the value obtained for wild-type LolB and shown as the mean ± standard deviation for triplicate experiments. (B) Close-up view of LolA R43L-LolB structure highlighting the two clusters of charged residues R34 and R61 (RR) and R175, K177 and K179 (RKK) on the convex side of the LolB β-barrel. (C) Transfer of Pal lipoprotein from LolA to LolB was assessed as described in (A) for wild-type LolB or variants containing alanine or glutamate mutations in the two charged clusters RR and RKK. (D) Expression of plasmid-borne wild-type lolB or variants in BW65 cells supported by expression of chromosomal lolB (top). Serial dilutions of a conditional lolB knockout E. coli BW65 strain carrying either plasmid-borne wild-type lolB or indicated variants in the absence of inducer required for expression of chromosomal lolB (bottom). (E) Structural alignment of lipoprotein-bound LolA (7Z6W) with the LolA R43L-LolB complex highlighting the steric clash of LolB Hook with the lipoprotein ligand. Acyl chain numbering is indicated. (F) Association with LolA and transfer of Pal lipoprotein from LolA to LolB as described in (A) for wild-type LolB or variants in the Hook. (G) Expression of LolB Hook mutants and their ability to support cell growth in the absence of chromosomal lolB evaluated as in (D). Controls for panels D and G demonstrating growth in the presence of chromosomally-encoded wild-type lolB can be found in SI Appendix Figure 2.

Key residues that control LolB helical lid release.

(A) Residues targeted for mutagenesis are shown as orange sticks in wild-type (WT) LolB (grey, 1IWM). (B) Binding of DAUDA to LolB wild-type and variants. The control corresponds to basal DAUDA fluorescence in the absence of protein. (C) Comparison of the structures of LolB WT (grey), and the Q44A mutant in the closed (light orange) and open (dark orange) conformation. Protein cavities are shown as solid teal surfaces. (D) Close-up views of the α2 helix (top left), the α2-α3 linker (top right) and the LolB central cavity (bottom).

Structure of lipoprotein-bound LolB and conformation rearrangements upon association with LolA and lipoprotein.

(A) Overall structure of LolB L114G (yellow) complexed with lipoprotein (cyan, stick representation). The mesh represents the Polder omit map of the lipoprotein ligand contoured at 3 σ. Positions of the lipoprotein acyl chains adopted in the two LolB chains within the asymmetric unit are displayed in green inset. (B) Cross section views of the LolB cavity cut at the points indicated in (A), displaying residues which interact with the lipoprotein acyl chains. (C) The LolB cavity visualized as a solid cyan cast in the LolB-lipoprotein and in the LolA-lipoprotein structure (7Z6W) with approximate volume indicated. (D) Alignment of LolB structures in its free (1IWM, light grey), LolA-bound (33DL, yellow) and lipoprotein-associated (33DM, orange) form. Arrows are highlighting movement adopted by indicated regions from the free to LolA-bound state 1) and from the latter to the lipoprotein-associated confirmation 2). The lipoprotein is shown in cyan. (E) Rmsd plots of residues from LolB in the free-state (1IWM) to LolA-bound (33DL, light grey) or to lipoprotein-associated conformation (33DM, yellow). LolB protein secondary structural elements are shown underneath.

Model of lipoprotein transfer from LolA to LolB and subsequent membrane insertion.

1) Lipoprotein receptor LolB (yellow) can associate with the membrane in either a ‘Hook-in’ or ‘Hook-out’ conformation. The LolB Belt and Hook are highlighted in green and red, respectively. 2) LolA-lipoprotein complex approaches LolB. 3) In the ‘Hook-out’ conformation, LolB engages the LolA-lipoprotein complex via Belt residues, releasing the ‘latch’ of the LolB cavity entrance. Insertion of the LolB Hook into the LolA cavity initiates transfer of the lipoprotein acyl chains (inset). 4) Following transfer of the acyl chains into LolB, the protein rotates to a ‘Hook-in’ conformation. 5) LolA is released, and LolB initiates the insertion of the lipoprotein acyl chains into the membrane. 6) The lipoprotein is fully integrated into the outer membrane, leaving LolB free to engage a new LolA-lipoprotein complex.

Comparison of E. coli LolA R43L-LolB structure with published crosslinking and structural data.

(A) LolA and LolB in vivo photo-crosslinking results (40) are mapped onto our LolA R43L-LolB structure. Residues which formed photo-inducible crosslinks when replaced with pBPA (p-benzoyl-L-phenylalanine) are shown in red, whereas residues unable to crosslink are in blue. (B) Alignments of E. coli LolA R43L-LolB (Ec) with the published X. campestris (Xc) LolA-LolB structure (8ORN) (42). (C) Close-up views of LolB Hook and Belt residues in the E. coli and X. campestris structures. (D) Residues located at the protein-protein interface in the LolA-LolB structures are shown as spheres (E. coli 33DL and X. campestris 8ORN).

Control plates for the in vivo characterization of LolB Belt and Hook mutants.

(A) Serial dilutions of a conditional lolB knockout E. coli strain (BW65) carrying plasmid-borne wild-type lolB or indicated variants in the R34/R61 (RR) or R175/K177/K179 (RKK) Belt cluster. Cells were grown in the presence of 0.2% arabinose to induce expression of chromosomally encoded wild-type lolB. The plate is a control for the experiment shown in Figure 3D. (B) Serial dilutions of E. coli BW65 carrying plasmid-borne wild-type lolB or indicated Hook variants in the presence of 0.2% arabinose to induce expression of chromosomally encoded wild-type lolB. The plate is a control for the experiment shown in Figure 3G.

Characterization of the LolB L114G mutant.

(A) His-tagged LolB wild-type or L114G variant were incubated with untagged LolA-Pal complexes. LolB and associated Pal were isolated on Ni resin and the elution fraction loaded onto SDS-PAGE. The amount of Pal transferred to LolB is reported, normalized to the value for wild-type LolB. Data shown are mean values ± standard deviation from triplicate experiments. (B) Serial dilutions of a conditional lolB knockout E. coli strain carrying either plasmid-borne wild-type lolB or L114G variant in the absence (left) or presence (right) of inducer required for expression of chromosomal lolB (top). Immunoblot showing the expression of plasmid-borne wild-type lolB and L114G variant in E. coli supported by growth of chromosomal lolB (bottom).

Structural comparison of LolB L114G in its free, LolA-bound and lipoprotein-associated form.

(A) Superimposition of the two lipoprotein ligands present in the asymmetric unit of the lipoprotein-bound LolB L114G crystal. The position of G114 Cα is indicated by a yellow sphere. The native L114, modeled in the lipoprotein-bound structure, is shown as orange transparent spheres. (B) Major conformational changes of LolB from its free form to the LolA- and lipoprotein-associated state. Circles represent close-up views of the indicated regions of LolB (left, 1IWM) in each state. A molecular morph highlighting the LolB transition between these three states is shown in Movie S3. (C) The residue-by-residue rmsd between free and lipoprotein-associated LolA (1UA8, 7Z6W) or LolB (1IWM, 33DM) is displayed on the lipoprotein-bound LolA (7Z6W, blue) or LolB (33DM, yellow) structures. Rmsd values (Å) per aligned Cα atom are shown as a continuous gradient from blue or yellow to red with a maximal deviation of 8 Å. Distance between the C-terminus of α1 helix at the bottom of the cavity and the closest lipoprotein atom is indicated, together with the relative lipoprotein position in the LolA and LolB structures. (D) Alignment of lipoprotein-bound LolA (7Z6W, blue) and LolB (33DM, yellow) onto our LolA-LolB structure. The distance between the invariant +1 cysteine from each lipoprotein (cyan) is indicated.

Comparison of simulated states of LolB on the membrane and their superimposition with LolA- and lipoprotein-bound structures.

(A) Molecular dynamics derived ‘Hook-in’ and ‘Hook-out’ models of LolB associated with the membrane (45). LolB Hook is in red and residues from the Belt are represented as green sticks. The acyl chains of LolB are shown in black. (B) Superimposition of the LolA R43L-LolB (left) and lipoprotein-bound LolB L114G (right) structures with the ‘Hook-in’ and ‘Hook-out’ models of LolB derived from molecular dynamics simulations. The lipoprotein is shown in cyan with its approximate distance to the membrane indicated.

X-ray data and refinement statistics.

List of primers for PCR amplification.

List of plasmids.