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Characterization of interactions and phospholipid transfer between substrate binding proteins of the OmpC-Mla system Bilge Ercan, Wen-Yi Low, Xuejun Liu, and Shu-Sin Chng Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00897 • Publication Date (Web): 04 Oct 2018 Downloaded from http://pubs.acs.org on October 5, 2018
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Biochemistry
Characterization of interactions and phospholipid transfer between substrate binding proteins of the OmpC-Mla system Bilge Ercana,‡, Wen-Yi Lowa,‡, Xuejun Liub, Shu-Sin Chnga,c,* a
Department of Chemistry, National University of Singapore, Singapore 117543. School of Pharmacy, University College London, United Kingdom. c Singapore Center for Environmental Life Sciences Engineering (SCELSE-NUS), Singapore 117456. b
Abstract: The outer membrane (OM) of Gram-negative bacteria is a permeability barrier that impedes the entry of external insults, such as antibiotics and bile salts. This barrier function depends critically on the asymmetric lipid distribution across the bilayer, with lipopolysaccharides (LPS) facing outside and phospholipids (PLs) facing inside. In Escherichia coli, the OmpC-Mla system is believed to maintain OM lipid asymmetry by removing surface exposed PLs and shuttling them back to the inner membrane (IM). How proteins in the pathway interact to mediate PL transport across the periplasm is not known. Evidence for direct transfer of PLs between these proteins is also lacking. In this study, we mapped the interaction surfaces between the two PL-binding proteins, MlaC and MlaD, using site-specific in vivo photocrosslinking, and obtained a physical picture for how these proteins may transfer PLs. Furthermore, we demonstrated using purified proteins that MlaD spontaneously transfers PLs to MlaC, suggesting that the latter has a higher affinity for PLs. Our work provides insights into the mechanism of bacterial inter-membrane lipid transport important for the maintenance of OM lipid asymmetry.
The Gram-negative cell envelope has two lipid bilayers with entirely different architectures. Immediately surrounding the cytoplasm is the inner membrane (IM), a typical phospholipid (PL) bilayer. The outer membrane (OM), separated from the IM by an aqueous periplasm, is an asymmetric lipid bilayer where PLs and lipopolysaccharides (LPS) reside in the inner and outer leaflets, respectively.1,2 LPS molecules pack densely in the presence of divalent cations to form a continuum; this outer layer serves as a barrier with significantly reduced permeability to antibiotics and other insults,3 contributing in part to why Gram-negative bacterial pathogens are difficult to kill. Lipid asymmetry at the OM is established via direct placement of LPS into the outer leaflet, and PLs into the inner leaflet. LPS transport is mediated by the Lpt machinery.4 PL transport to the OM is not yet understood. During growth, small amounts of PLs may end up in the outer leaflet of the OM, causing perturbations in lipid asymmetry. These defects can be exacerbated under stress, such as during exposure to divalent cation chelators or when OM biogenesis is disrupted.5,6 The continuous accumulation of PLs in the outer leaflet of the OM creates regional PL bilayer patches that would compromise its barrier function.3 Therefore, there are systems in cells evolved to ensure that lipid asymmetry at the OM is always preserved. OmpLA and PagP are two enzymes that modify PLs in the outer leaflet of the OM. OmpLA hydrolyzes acyl chains of outer leaflet PLs,7 and PagP acylates LPS8 or phosphatidylglycerol (PG)9 using outer leaflet PLs as substrates.
Both proteins act on mislocalized PLs to modify PL and LPS structures, alleviating adverse effects on OM lipid asymmetry. The OmpC-Mla system is also important for the maintenance of lipid asymmetry at the OM; removing any member of this system results in PL accumulation in the outer leaflet.10,11 However, in contrast to OmpLA and PagP, it is proposed to do so by removing PLs from the outer leaflet of the OM and transporting them back to the IM.10 The OmpC-Mla system is composed of seven proteins located across the cell envelope: the OmpC-MlaA complex at the OM, MlaC in the periplasm, and the MlaFEDB complex at the IM. PLs are believed to be extracted from the outer leaflet of the OM by the OmpC-MlaA complex, passed along to MlaC, and finally delivered to the IM via the MlaFEDB complex.10,11 This directionality of PL transport has recently been demonstrated in E. coli, where overexpression of MlaC and the IM complex partially rescues cells with defects in retrograde (OM-to-IM) transport of bulk PLs.12 How the OmpC-Mla system transports PLs is not known, but studies on individual subcomplexes have revealed interesting insights into the process. Recent structural13 and biochemical14 characterization of the OmpC-MlaA complex established that MlaA forms a membrane channel adjacent to OmpC, and likely provides a route for PL translocation across the OM. MlaC is a lipid chaperone and has been crystallized with a PL bound in a deep hydrophobic pocket;15,16 molecular dynamics (MD) simulations suggest that this pocket exhibits high conformational flexibility, allowing it to accommodate the ligand during binding.17 The IM MlaFEDB complex represents a noncanonical ATPbinding cassette (ABC) transporter, where MlaB and MlaD perform functions auxiliary to the core MlaFE complex.18 The cytoplasmic protein MlaB has vital roles in stabilizing the MlaFEDB complex and regulating its ATP hydrolytic activity. Within the complex, MlaD forms stable hexamers via its periplasmic domain (sMlaD),18 exhibiting a donut-shaped architecture containing a central hydrophobic pore.16 Interestingly, MlaD also binds PLs,16,18 suggesting that PL transfer from MlaC to the IM occurs via MlaD. To gain mechanistic insights into PL transport mediated by the OmpC-Mla system, we focused on MlaC. Consistent with its proposed role in shuttling PLs from the OM to the IM, it has been shown that MlaC can interact with MlaA-porin and MlaFEDB complexes in vitro.16 However, it is unclear whether these interactions exist in cells, and more importantly, how exactly MlaC interacts with these complexes to transport PLs. Therefore, we sought to map the interaction surface(s) between MlaC and other OmpC-Mla components within cells. Guided by the crystal structure of the ortholog in Ralstonia solanacearum,15 we replaced 37 residues distributed around the surface of MlaC with para-benzoyl-L-phenylalanine (pBpa) and looked for UV-
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activatable crosslinking to other OmpC-Mla components. This technique allows us to covalently link interacting partners in cells and is especially useful for trapping transient interactions.19 We found one position (V171) that allowed strong UV-crosslinking of MlaC (MlaC-His ~23 kDa) to two other proteins (Figure 1A). One of these proteins is MlaA (~26 kDa), as the crosslinked adduct (top band, ~50 kDa) was detected by an α-MlaA antibody. To identify the other interacting partner, we performed affinity purification of the crosslinked adducts and analyzed them using tandem mass spectrometry (MS/MS). Both crosslinked adducts were found in the membrane fraction, indicating that the second interacting partner of MlaC is also a membrane protein (Figure S1A). MS/MS analysis confirmed the identity of the top crosslinked adduct as MlaA. Interestingly, crosslinking of MlaC to MlaA also allowed it to pull down a ~37 kDa protein that exhibits heatmodifiable gel shift, suggesting MlaC interacts with MlaA in complex with porins (OmpC/F).11 MS/MS analysis further revealed that the bottom crosslinked adduct (~45 kDa) is MlaD (~20 kDa) (Figure 1B). Crosslinking of MlaC to MlaD allowed it to pull down non-crosslinked MlaD, consistent with the idea that MlaC interacts with MlaD in its hexameric form.18 Both crosslinked and non-crosslinked bands of MlaD no longer appeared in cells lacking MlaD (Figure S1B), validating these findings. We conclude that MlaC interacts with MlaA and MlaD in cells as part of their native complexes.
Figure 1. MlaC interacts with MlaA and MlaD via different but overlapping surfaces in vivo. (A) Representative immunoblots showing UVcrosslinked bands formed in ∆mlaC cells expressing MlaC-His substituted with pBpa at indicated positions. (B) Co-TALON affinity purification of crosslinked adducts of MlaCV171pBpa-His. MS/MS sequencing confirmed the identities of crosslinked adducts as (1) MlaCV171pBpa-MlaA and (2) MlaCV171pBpa-MlaD. The top three most abundant proteins detected in each band are tabulated. (C, D) Immunoblots showing the presence or absence
of UV-crosslinked bands (open arrowheads) formed in ∆mlaC, ∆mlaA (C) or ∆mlaD (D) cells expressing MlaC-His substituted with pBpa at positions close to V171. Unknown crosslinked adducts are indicated by asterisks. Unless otherwise stated, samples were heated before SDS-PAGE analysis and visualized by silver staining and/or immunoblots using α-His and α-MlaA antibodies. (E) Cartoon representation of the crystal structure of E. coli MlaC with a bound PL molecule (PDB: 5UWA)16 with positions that allow UV-crosslinking to MlaA, MlaD, or both, indicated as yellow, red, and green sticks, respectively. Positions selected in the initial and localized search but did not give crosslinking are indicated in light green and white, respectively.
MlaC contacts both MlaA and MlaD through residue V171. To demarcate MlaC-MlaA and MlaC-MlaD interaction surface(s), we generated an additional 17 pBpa-containing MlaC variants, focusing on residues near V171. Five of these positions enabled UVcrosslinking to MlaA (albeit weaker than V171) (Figure 1C); while these adducts were not detected by the α-MlaA antibody (possibly because of blocked epitope), they were absent in ∆mlaA cells. Three residues allowed the formation of MlaD-crosslinked adducts, which were correspondingly no longer observed in cells lacking MlaD (Figure 1D). We showed that these MlaCpBpa variants that allowed crosslinking can functionally replace wildtype MlaC (Figure S2A). The MlaA- and MlaD-crosslinking positions map to adjacent regions on one face of MlaC from E. coli (Figure 1E), whose structure has recently been solved.16 We conclude that MlaC interacts with MlaA and MlaD via different but overlapping surfaces. Using photo-crosslinking in cells, we have established where MlaD binds on MlaC. To characterize where MlaC binds on MlaD, we used the same strategy and incorporated pBpa into multiple locations on MlaD. The crystal structure of sMlaD reveals a donut-shaped hexamer.16 Four out of 23 positions on MlaD, when replaced with pBpa, gave UV-crosslinked bands in WT cells (Figure 2A). These bands could be detected by the αMlaC antibody, indicating the presence of MlaDpBpa-MlaC adducts. All these positions contacting MlaC were found on the periplasm-facing side of MlaD hexamers (Figure 2B). Interestingly, all crosslinking positions localize across the interface between two MlaD monomers in the hexameric structure, delineating potential sites for MlaC interaction. The interfacial positions of these residues could also allow formation of MlaDpBpa-MlaD crosslinks. Consistent with this idea, UV-crosslinked bands were still observed in cells lacking MlaC, and the intensities of these bands were reduced in ∆mlaD cells (Figure 2A). Removing endogenous MlaD additionally affected the levels of MlaDpBpa-MlaC crosslinks. This observation is not fully understood but we showed that these MlaDpBpa variants are only partially functional (Figure S2B); mutations at the MlaD-MlaD interface likely impacted on the stability and/or function of pure MlaDpBpa or mixed MlaDpBpaMlaDWT hexamers, thereby affecting interactions with MlaC. Taken together, our results in part suggest why MlaD oligomerizes in the IM complex and may have implications for how PLs can be transferred between MlaC and MlaD. We have now established that MlaC and MlaD interact with each other in cells. Others have shown that MlaC can interact with the MlaFEDB complex in vitro,16 likely through MlaD. However, whether these interactions are functional for PL transfer between MlaC and MlaD is unclear. To address this question, we sought to reconstitute PL transfer using purified proteins. We have previously demonstrated that overexpressed sMlaD co-purifies with endogenous PLs.18 Similar to sMlaD, we found that natively purified MlaC also contained PLs (Figure S3A), explaining why MlaC can be crystallized with PL bound.15,16 To enable PL transfers in vitro, we also generated PL-free (apo) forms of MlaC and sMlaD. These proteins were purified under denaturing conditions, washed extensively, and refolded to give preparations lacking PLs (Figure S4A). Both refolded MlaC and sMlaD exhibited similar size exclusion chromatographic (SEC) profiles
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Biochemistry to natively purified MlaC monomer and sMlaD hexamers, respectively (Figure S4B), indicating that the apo proteins are properly folded.
Figure 2. MlaC binds at the dimeric interface(s) of MlaD hexamers. (A) Representative immunoblots showing MlaDpBpa-MlaC and MlaDpBpa-MlaD UV-crosslinked bands (open arrowheads) formed in WT, ∆mlaC and ∆mlaD cells expressing MlaD-His substituted with pBpa at indicated positions. Samples were heated before SDS-PAGE analysis and immunoblots performed using α-His and α-MlaC antibodies. (B) Side (left) and top (right) views of the cartoon representation of the crystal structure of sMlaD from E. coli (PDB: 5UW2)16 with positions that allow UVcrosslinking to MlaC (and MlaD) indicated as orange sticks. Adjacent monomers in the hexamer are highlighted in different shades of blue.
We first tested whether purified MlaC and sMlaD can interact in a specific manner by recapitulating pBpa-dependent photocrosslinking in vitro. We overexpressed and purified MlaC containing pBpa at position V171, which allowed photocrosslinking to MlaD in cells (Figure 1B). MlaCV60pBpa was used as a negative control. Both pBpa variants were purified to homogeneity (Figure S3B) and incubated with purified sMlaD hexamers. Upon UV-irradiation, we observed crosslinked adducts between sMlaD and MlaCV171pBpa, but not MlaCV60pBpa (Figure S3C), demonstrating that MlaC-sMlaD interactions in vitro likely occur in a manner similar to that observed in cells, and is thus specific. Current evidence indicates that the OmpC-Mla pathway is a retrograde PL transport system.10,12 We therefore set up initial assays to monitor PL transfer from MlaC to sMlaD. We incubated PL-bound (holo) MlaC with apo sMlaD, separated them using SEC (with minimal cross-contamination, Figure S5), and analyzed organic extracts on thin layer chromatography (TLC). Surprisingly, we found that PLs were still associated with MlaC, indicating little/no transfer to sMlaD (Figure 3A). Instead, the same experiments conducted with holo sMlaD and apo MlaC revealed that PLs could be spontaneously transferred from sMlaD to MlaC (Figure 3B). Overall, these results indicate that MlaC has higher binding affinity for PLs than sMlaD, but also bring into question the true directionality of PL transport in the OmpC-Mla system.
Figure 3. sMlaD spontaneously transfers PLs to MlaC in vitro. (A) TLC analysis of PLs extracted from holo MlaC-His and apo sMlaD-His before (marked by asterisks) and after incubation with each other (bolded/ marked by hashes). (B) TLC analysis of PLs extracted from apo MlaC-His and holo sMlaD-His before (marked by asterisks) and after incubation with each other (bolded/marked by hashes). CL; cardiolipin, PE; phosphatidylethanolamine, PG; phosphatidylglycerol. (C) A proposed model for how the ability of MlaC in binding PLs at high affinity explains possible energetic requirements within the OmpC-Mla pathway. (D) Top and bottom views of the surface mesh representation of sMlaD from E. coli (PDB: 5UW2)16 illustrating cavities (black) radiating out from the central hydrophobic pore (red) along the interfaces between MlaD monomers.
Spontaneous PL transfer from sMlaD to MlaC in vitro highlights the distinct possibility that the OmpC-Mla system may in fact transport PLs in an anterograde (IM-to-OM) fashion. However, this observation can also be interpreted in the context of retrograde PL transport, which is strongly supported by genetic evidence.10,20 Homologs of the Mla IM complex have also all been characterized as lipid uptake systems either in chloroplasts or actinobacteria.21–23 Furthermore, we have previously demonstrated that overexpression of MlaFEDB together with MlaC can rescue defects in retrograde PL transport observed in cells lacking the Tol-Pal complex.12 We believe that high-affinity binding of PLs to MlaC can help explain the energetic requirements, or lack of, at each step of retrograde PL transport mediated by the OmpCMla system. Specifically, strong PL binding may drive OmpCMlaA-facilitated PL transfer from the outer leaflet of the OM to MlaC in the absence of energy input (Figure 3C). Subsequently, because PLs are bound tightly to MlaC, ATP hydrolysis by the MlaFEDB complex may be needed to release these lipids from MlaC, and only then allow transfer to MlaD and eventually into the IM. Such ATP-dependent release of cargo from substrate binding proteins is reminiscent of ABC importers,24 as exemplified by the maltose transporter.25 Our crosslinking experiments shed light on how MlaC and MlaD interact to allow PL transfer. Both MlaA and MlaD contact MlaC near the entrance of the PL-binding cavity, likely reflecting
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functional interactions. Interestingly, MlaC contacts the MlaD hexamer along one or more of its dimeric interfaces, where it can be positioned to deliver PLs into the central hydrophobic pore observed in the hexameric structure (Figure 3D). It is likely that the acyl tails of a PL molecule could bind to this pore as it makes its way across the MlaD hexamer towards the IM; however, it is less clear where the polar headgroup would bind. Since MlaC interacts in the region between two MlaD monomers, we speculate that the headgroup of a transported PL molecule traverses MlaD through these interfaces. In support of this idea, we noted that there are continuous cavities radiating out from the central hydrophobic pore along each dimeric interface of the MlaD hexamer (Figure 3D), representing sites where PL headgroups could bind. While this hypothesis needs to be thoroughly investigated, we now have a working model for how MlaC and MlaD transfer PLs. This physical picture would eventually be useful as we work towards understanding PL transfer between MlaC and the full MlaFEDB complex. Proper maintenance of lipid asymmetry is required for the OM to serve as an effective permeability barrier. This makes the OmpC-Mla system an attractive target for antibiotic intervention against Gram-negative infections. Further to improving our understanding of bacterial PL transport, our work towards deciphering molecular mechanisms within the OmpC-Mla system, and the assays developed herein, would possibly help in future identification and/or validation of small molecule inhibitors.
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Materials and Methods, Figures S1-S5 (PDF)
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AUTHOR INFORMATION (21)
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‡These authors contributed equally.
Funding Sources B.E. was supported by the Singapore International Graduate Award (SINGA). W.-Y.L. was supported by the NUS President’s Graduate Fellowship (PGF). This work was supported by the NUS Start-up funding, the Singapore Ministry of Education Academic Research Fund Tier 1 and Tier 2 (MOE2013-T2-1-148) grants (to S.-S.C.).
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Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT We thank Ross Tomaino (Taplin MS Facility, Harvard Medical School) for MS/MS analyses. We thank Rahul Shrivastava, KangWei Tan and Zhi-Soon Chong for providing expression plasmids. We acknowledge Moses Thien for help in initial experiments.
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Biochemistry
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