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Cryo-EM structures of LolCDE reveal the molecular mechanism of bacterial lipoprotein sorting in Escherichia coli [1]

['Weiwei Bei', 'National Laboratory Of Biomacromolecules', 'Institute Of Biophysics', 'Chinese Academy Of Sciences', 'Beijing', 'University Of Chinese Academy Of Sciences', 'Qingshan Luo', 'Huigang Shi', 'Haizhen Zhou', 'Min Zhou']

Date: 2022-10

Bacterial lipoproteins perform a diverse array of functions including bacterial envelope biogenesis and microbe–host interactions. Lipoproteins in gram-negative bacteria are sorted to the outer membrane (OM) via the localization of lipoproteins (Lol) export pathway. The ATP-binding cassette (ABC) transporter LolCDE initiates the Lol pathway by selectively extracting and transporting lipoproteins for trafficking. Here, we report cryo-EM structures of LolCDE in apo, lipoprotein-bound, and AMPPNP-bound states at a resolution of 3.5 to 4.2 Å. Structure-based disulfide crosslinking, photo-crosslinking, and functional complementation assay verify the apo-state structure and reveal the molecular details regarding substrate selectivity and substrate entry route. Our studies snapshot 3 functional states of LolCDE in a transport cycle, providing deep insights into the mechanisms that underlie LolCDE-mediated lipoprotein sorting in E. coli.

Funding: This work was supported by grants from the National Natural Science Foundation of China (31625009 to Y.H. and 21575065 to X.Z.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37020201 to Y.H.), the Ministry of Science and Technology (2016YFA0500404 to Y.H.) and the China Postdoctoral Science Foundation (BX20190356 to Q.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Density maps of the apo-LolCDE, RcsF-LolCDE and AMPPNP-LolCDE complexes are available through the EMDB with entry codes EMD-31804, EMD-31803 and EMD-31802, respectively. Coordinates of the apo-LolCDE, RcsF-LolCDE and AMPPNP-LolCDE complexes are deposited in the Protein Data Bank (PDB) with the accession codes 7V8M, 7V8L and 7V8I, respectively.

Importantly, LolCDE belongs to the ABC3 superfamily of ABC transporters, also known as type VII ABC transporters [ 32 ], does not transport substrates across the IM, rather they selectively extract mature lipoproteins from the outer leaflet of the IM and transfers them to LolA, propelled by cytoplasmic ATP hydrolysis. This is in stark contrast to canonical ABC transporter. Recently, Kaplan and colleagues reported the crystal structure of LolA in complex with the periplasmic domain of the LolC [ 25 ]. Tang and colleagues and Sharma and colleagues determined cryo-EM structures of LolCDE in different conformational states [ 33 , 34 ]. High-resolution structures of the Lol components promise to greatly advance our understanding of the molecular mechanism of lipoprotein biogenesis. Here, we reported cryo-EM structures of LolCDE in its apo, RcsF- and AMPPNP-bound states. We point out that the apo-LolCDE structure we obtained is strikingly different from what was previously reported [ 33 ]. Furthermore, our structure-based functional analysis reveals a clear path through which lipoproteins enter the substrate-binding cavity of LolCDE.

( 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. Mg 2+ 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, Loop LolC and Loop LolE . ( 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.

The outer membrane (OM), hallmark of gram-negative bacteria, lies at the frontline of interaction with environment serving as a potent permeability barrier that prevents entry of many toxic substances into the cell [ 1 , 2 ]. Central to OM biogenesis and physiology are the lipoproteins that peripherally anchored to the membrane via their N-terminal lipid moiety. OM lipoproteins underpin the functioning of a diverse array of machineries that are responsible for the lipopolysaccharides (LPS) export, assembly of integral OM proteins and peptidoglycan cell wall, to name a few [ 3 – 6 ]. Lipoproteins are therefore indispensable for the survival of gram-negative bacteria [ 7 ], bearing important implications for efforts to develop novel antimicrobial agents against multidrug-resistant bacteria [ 8 – 12 ]. In gram-negative bacteria, lipoproteins are synthesized in the cytoplasm and matured in the inner membrane (IM). In Escherichia coli, maturation of lipoproteins occurs on the periplasmic face of the IM and involves consecutive modifications by 3 membrane-bound enzymes Lgt, Lsp, and Lnt [ 13 – 18 ]. Thereafter, they are either retained in the IM or transferred to the inner leaflet of the OM, the determinant being the presence of Asp at +2 position followed by certain residues at +3, the so-called Lol avoidance signal [ 19 , 20 ]. Lipoproteins with the signal remain in the IM, whereas others enter the Lol pathway for transport to the OM [ 21 , 22 ]. The Lol pathway comprises 5 Lol proteins, LolA-E [ 7 , 20 ] ( Fig 1A ). Among them, LolCDE, an IM-embedded ATP-binding cassette (ABC) transporter initiates lipoprotein sorting by selectively extracting OM-destined lipoproteins from the outer leaflet of the IM [ 23 ] and transfers them to LolA [ 24 , 25 ], a periplasmic chaperone. LolB, itself an OM-localized lipoprotein, accepts LolA-bound lipoproteins in a “mouth-to-mouth” manner and inserts them into the inner leaflet of the OM via their N-terminal acyl chains [ 26 – 29 ]. At least 90 different lipoproteins have been identified as substrates of the Lol pathway in E. coli, and they perform a diverse array of functions after being sorted to their final destinations [ 30 , 31 ].

Results

Verification of the apo-LolCDE structure In our apo-LolCDE structure, the Cα-Cα distance between Ala106 in PLDLolC and Ser173 in PLDLolE is only 5.4 Å, whereas the distance is increased to 48.6 Å in apo-LolCDE* [33] (Fig 2A). To validate our structure, 2 cysteines were incorporated into LolCDE to generate LolCA106CDES173C mutant in order to probe the distance between LolC and LoE in LolCDE. In the absence of reducing reagents, we observed intermolecular disulfide bond between LolCA106C and LolES173C almost fully formed in the purified LolCA106CDES173C protein. Addition of β-mercaptoethanol (β-ME) disrupted the intermolecular disulfide bond (Fig 2B). We thus conclude that the arrangement of the 2 PLDs in LolCDE agrees well with our apo-LolCDE structure. Furthermore, the crosslinks remained the same, regardless of the presence of RcsF (Fig 2B). These results further confirmed that both the apo- and RcsF-bound LolCDE adopt the same conformations as we observed. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3001823.g002 To investigate whether conformational changes of apo-LolCDE are required for RcsF entry into the V-shaped cavity, we incorporated a photo-crosslinkable unnatural amino acid (p-benzoyl-phenylalanine (pBPA)) [43] at Leu256 of LolC (Fig 2A), whose side chain points to the V-shaped cavity. As shown in Fig 2C, LolC×RcsF adducts are detected when both RcsF and LolCDE were reconstituted in nanodisc, demonstrating that the exogenously added RcsF is able to enter the V-shaped cavity of LolCL256pBPADE (no Cys mutations). Furthermore, we also detected the LolE-LolC×RcsF adducts in LolCA106CDES173C sample upon UV radiation. This observation implicates that conformational change of the 2 PLDs of LolC and LolE is not required for the entry of RcsF into the V-shaped cavity (Fig 2C). We observed, however, post-RcsF entry into the V-shaped cavity of disulfide-bonded LolCA106CDES173C complex; it failed to release from the complex to LolA, unless the intermolecular disulfide bond is disrupted by the addition of reducing reagent (Fig 2D). Failure of RcsF release to LolA by the LolCA106CDES173C complex suggests that either an overall conformational change of LolCDE is required or the disulfide bond-fixed PLDs interfere with the lipoprotein transport. In line with our in vitro photo-crosslinking findings, complementation assay also showed that lolCA106CDES173C failed to rescue the growth of the lolCDE-depleted E. coli cells unless reducing reagent is supplemented (Figs 2E and S10A). Taken together, we conclude that our apo-LolCDE structure represents the correct apo conformational state of LolCDE, which resembles closely the conformation of RcsF-LolCDE. This study also correlates well with the structural and functional studies of LptB 2 FG [39–42].

RcsF-LolCDE interactions and lipoprotein substrate selectivity In our RcsF-LolCDE structure, 3 acyl chains (R1, R2, and R3) and the N-terminal 14 residues of the mature RcsF are well resolved (the first Cys residue of a mature lipoprotein named +1 position) (Fig 3A). Specifically, R1 sits between TM1LolC and TM2LolE (Fig 3B) by making hydrophobic interactions with residues Val44, Val47, and Met48 of TM1LolC and Met267 of TM2LolE (Fig 3C). In the opposite side, R2 and R3, surrounded by TM2LolC, TM1LolE, and LoopLolE (Fig 3B), are in close contact with residues Met266 and Met267 of TM2LolC, Val43, and Phe51 of TM1LolE, as well as Met261, Ile265, and Ile268 of TM2LolE (Fig 3C). To probe the functional significance of these contacts, we made point mutations for the abovementioned residues by introduction of hydrophilic residues. We then tested these mutants one by one. Each LolCDE mutant is successfully expressed and assembled giving the right size exclusion chromatography profile (S11A Fig). These mutations (the sole exception being M267D of TM2LolE), however, all have their substrate entry severely affected, as inferred from no detectable LolC×RcsF adducts by photo-crosslinking (Fig 3D). Consistent with this, complementation assays also showed that these lolCDE mutants supplement failed to fully restore the growth of the lolCDE-depleted E. coli to a varied degree (Figs 3E and S10B). These findings highlight the functional importance of 3 acyl chain-interacting residues in LolCDE and, in turn, corroborate our structural observations. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3001823.g003 Of the 14 visible residues of RcsF in the RcsF-LolCDE structure, only 3 residues, Cys+1, Ser+2, and Met+3, are enclosed by the V-shaped cavity of LolCDE (S13A Fig). The remaining residues adopt a stretched loop conformation protruding from the cavity on the LoopLolC side (Fig 3B). First, we tested the functional importance of Met+3-interacting residues in LolC. As shown in Fig 3G, we found that mutations (M48D, F51D, L55D, and V260D of LolC) did not affect the complex assembly (S11B Fig). However, they severely interfered with the entry of RcsF and failed to rescue the growth of the lolCDE-depleted E. coli (Figs 3H and S10C). Next, we tested whether our structural observations agree well with the Lol avoidance signal hypothesis. In line with the hypothesis, we found that S21D (+2 position), S21E (+2 position), as well as a series of hydrophobic-to-hydrophilic mutations at the +3 position of RcsF either abolished or severely affected RcsF entry into the V-shaped cavity of LolCDE (Fig 3I). Taken together, our structural observations and functional studies reveal a bipartite binding mode of a lipoprotein to LolCDE. The 3 acyl chains and the first 3 residues of a mature lipoprotein dictate its binding affinity to LolCDE and substrate selectivity, respectively, thereby providing a structural explanation for the Lol avoidance signal hypothesis.

A single path for lipoprotein entry into the substrate-binding cavity As mentioned above, the substrate-binding cavity of apo-LolCDE has 2 intermolecular interfaces, the LoopLolC-TM2LolE interface (denoted as Interface I) and the LoopLolE-TM2LolC interface (denoted as Interface II) (Fig 6A), which can both serve as potential gates for lipoprotein entry. To figure out the exact entry route, we introduced intermolecular disulfide bonds to block 1 gate and probed the entry of RcsF via the other. In each case, however, we obtained only partial formation of disulfide bonds in the LolCDE samples; we therefore proceed focusing on the crosslinking of RscF and LolC-LolE, those crosslinks that do contain intermolecular disulfide bonds between LolC and LolE. A potential disulfide bond between E255CLolC and S362CLolE in LolCDE was first introduced in hope to prevent RcsF entry via the Interface II, and SDS-PAGE analysis revealed that, though not complete, disulfide bonds were formed in the LolCL256pBPA+E255CDES362C complexes (S13D Fig). After reconstitution into nanodisc together with Flag-tagged RcsF, the LolE-LolC×RcsF adducts were clearly detected under nonreducing condition upon UV radiation (Figs 6B and S16 and S1 Data). This demonstrated that formation of intermolecular disulfide bonds in the LolCDE complexes blocks the Interface II; however, the RcsF entry into the V-shaped cavity is unaffected. This strongly suggests that Interface I serves as the substrate entry route. Similarly, disulfide bond between L350CLolC and R263CLolE was introduced in hope to block Interface I. Again, intermolecular disulfide bonds formation was incomplete in the LolC L256pBPA+L350CDER263C complexes (S13D Fig), and no LolE-LolC×RcsF crosslinks were detected after in vitro photo-crosslinking (Fig 6B). Absence of LolE-LolC×RcsF crosslinks strongly implicates that RcsF is unable to enter the cavity with Interface I blocked. Base on the results, we propose that lipoproteins enter the cavity via Interface I rather than Interface II of LolCDE. These findings correlate well with our previous claim that the U-Loop in the LoopLolE takes part in buttressing the substrate-binding cavity in an outward conformation, and with the structural observation that the proteinaceous portion of RcsF is only located on the LoopLolC side in the RcsF-LolCDE structure. Results from our experiments are self-consistent and in line with structural observations. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3001823.g006

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