Fig 1.
Cryo-EM structures of LolCDE in different states.
(A) Schematics of the Lol pathway. Letters A to E designate LolA to LolE, respectively. (B) The 4.0-Å cryo-EM map of LolCDE. LolC, LolE and 2 copies of LolD are colored in blue, pink, yellow, and grey, respectively. The lipoprotein densities are shown in green. (C) Ribbon diagram of apo, RcsF-bound and AMPPNP-bound LolCDE structures. Mg2+ is shown in green spheres and AMPPNP in red sticks. (D) Cylindrical helix cartoon representation of structure motifs of apo-LolCDE. The U-Loop is highlighted in red. Dashed lines indicate the V-shaped cavity of LolCDE. (E) Top view of the V-shaped cavity, configured by TM1s and TM2s of LolC and LolE, LoopLolC and LoopLolE. (F-H) Conformational changes of LolCDE domains upon AMPPNP binding. (F) Top view of NBDs of RcsF-LolCDE (left) and AMPPNP-LolCDE (right). (G) Overlay of TMDs of RcsF-LolCDE (blue and pink) and AMPPNP-LolCDE (purple). The arrows indicate direction of TMs moving upon AMPPNP binding. The black and red dashed lines indicate the changes of the V-shaped cavity. (H) Overlay of PLDs of RcsF-LolCDE (blue and pink) and AMPPNP-LolCDE (purple). The arrows indicate directions of PLDs rotation upon AMPPNP binding.
Fig 2.
Verification of the apo-LolCDE structure.
(A) Comparison of our apo-LolCDE structure (left) to the apo-LolCDE* structure (right, PDB code:7ARI). Zoom-in view showing 2 amino acids in 2 PLDs, Ala106 and Ser173, which were replaced with cysteines in (B to E). Leu256LolC shown in purple spheres was substituted with pBPA for in vitro photo-crosslinking in (C). (B) Coomassie-stained SDS–PAGE gel assessing disulfide bond formation of LolCA106CDES173C and RcsF-LolCA106CDES173C. The samples of lanes 2 through 4 were supplemented with SDS loading dye without β-ME, and the samples of lanes 6 through 8 were supplemented with SDS loading dye with β-ME. Note that RcsF migrates slower after addition of reducing agent. (C) In vitro photo-crosslinking. LolCL256pBPADE proteins with or without 2 cysteine mutations were reconstituted with RcsF in nanodisc. The LolC×RcsF and the LolE-LolC×RcsF adducts were detected by immunoblotting. (D) The in vitro lipoprotein transport assays. To break intermolecular disulfide bond, the nanodisc-embedded RcsF-LolCA106CDES173C protein was incubated with TCEP prior to the addition of LolA (W70pBPA). (E) Complementation assays. The dilutions were spotted on LB plates with (right) or without (left) TCEP. Protein leaky expression levels of the LolC and LolE proteins were detected by western blotting (bottom). Data shown in (B to E) are representatives of 3 replicates.
Fig 3.
The bipartite binding mode between RcsF and LolCDE.
(A) Ribbon diagram of RcsF-LolCDE structure (right). Zoom-in view of the atomic model of RcsF superimposed with the cryo-EM densities (left). The arrow indicates the +1 position of the mature RcsF. The 3 acyl chains (R1, R2, and R3) and the N-terminal 14 residues of the proteinaceous portion are labelled. (B) Side view of the V-shaped cavity and the RcsF-binding mode. TM segments are shown in cylindrical helices. (C) Zoom-in view of the hydrophobic interactions between acyl chains (R1, R2, and R3) and LolC (left) or LolE (right). Residues shown as stick model were substituted with Asp for functional assays. (D) Photo-crosslinking assessing the importance of residues of LolCDE that interact with 3 acyl chains of RcsF in (C). rcsF were coexpressed with lolCDE mutants. (E) Complementation assay for the lolCDE mutants in (C). Protein leaky expression levels of the lolC and lolE mutants were detected by western blotting (bottom). (F) Zoom-in view of the hydrophobic interactions between Met+3 of RcsF and residues of LolCDE. (G and H) photo-crosslinking (G) and complementation assays (H) assessing the functional importance of hydrophobic interactions between Met+3 and residues of LolCDE. (I) UV-dependent crosslinks between LolCDE and RcsF variants were detected by immunoblotting. Data shown in (D and E) and (G to I) are representatives of 3 replicates.
Fig 4.
Functional importance of the negatively charged residues in the V-shaped cavity.
(A) Top view of TMDs of RcsF-LolCDE showing the negatively charged residues (red sticks) lining the upper interior of the V-shaped cavity. (B and C) The negatively charged residues in (A) were substituted with either Asn or Gln. Photo-crosslinking (B) and complementation assays (C) showing the critical roles of the 2 residues (Glu263LolCand Asp264LolE) for LolCDE function. (D) Cross-sectional view of the hydrophobic surface of the V-shaped cavity, showing the precise positioning of the RcsF into the cavity (left). Hydrophobic and hydrophilic regions of the substrate-binding cavity are colored in blue and red, respectively. Zoom-in view of the location of Cys+1 and Ser+2 of RcsF in the V-shaped cavity (right). The dashed lines and labels indicate the distances between the side chains of Glu263 and Asp264. (E and F) E263 and D264 were substituted with different types of amino acids for photo-crosslinking (E) and complementation assays (F), respectively. Data shown in (B, C, E, and F) are representatives of 3 replicates.
Fig 5.
The U-loop maintains the configuration of the substrate-binding cavity.
(A) Top view of the V-shaped cavity showing that the U-Loop (red) interacts with TMs. Residues from both TM1LolE and TM2LolC (in blue) make hydrophobic contacts with the U-Loop (red). (B and C) The 6 residues (S363-I368 of LolE) that consist of the U-Loop and the 7 residues (V349-A355 in LoopLolC) were deleted, respectively. Photo-crosslinking (B) and complementation assays (C) showing that the U-Loop is crucial for LolCDE function. (D and E) Residues (in red stick model) in (A) were substituted with Asp for photo-crosslinking (D) and complementation assays (E) respectively. Data shown in (B to E) are representatives of 3 replicates.
Fig 6.
A single path for lipoprotein entry into the V-shaped cavity and energy requirement for lipoprotein transfer to LolA.
(A) Top view of 2 potential gates (Interface I and Interface II) for lipoprotein entry (middle). Zoom-in view of 2 pairs of residues replaced with cysteines in (B). (B) In vitro photo-crosslinking. LolCL256pBPADE that contain 2 cysteine mutations or not were reconstituted with RcsF in nanodisc. The LolE-LolC×RcsF adducts were detected by immunoblotting. (C) LolCL256pBPADE that contain either wild-type LolD or LolD (E171Q) were reconstituted with RcsF in nanodisc. The adducts were evaluated by exposing to UV radiation with or without addition of ATP and Mg2+. (D) Scheme of an in vitro one-cycle lipoprotein transfer to LolA. Addition of LolA (W70pBPA), along with ATP and Mg2+, leads to transfer of RcsF from LolCDE to LolA. (E) Nanodisc-embedded RcsF-LolCDE proteins that contain either wild-type LolD or LolD (E171Q) were incubated with LolA (W70pBPA) and nucleotides. The ability to transfer RcsF to LolA (W70pBPA) from LolCDE was probed. Data shown in (B, C and E) are representatives of 3 replicates.
Fig 7.
Proposed model for lipoprotein extraction and transfer to LolA.
ATP and ADP are shown as balls in red and purple, respectively. The dashed arrow indicates the path for lipoproteins entry into the cavity. Our apo-LolCDE, RcsF-LolCDE and AMPPNP-LolCDE structures represent structures in ①, ②, and ③, respectively.