A dual substrate-accessing mechanism of a major facilitator superfamily protein facilitates lysophospholipid flipping across the cell membrane

Abstract

Lysophospholipid transporter (LplT) is a member of the major facilitator superfamily present in many Gram-negative bacteria. LplT catalyzes flipping of lysophospholipids (LPLs) across the bacterial inner membrane, playing an important role in bacterial membrane homeostasis. We previously reported that LplT promotes both uptake of exogenous LPLs and intramembranous LPL flipping across the bilayer. To gain mechanistic insight into this dual LPL-flipping activity, here we implemented a combination of computational approaches and LPL transport analyses to study LPL binding of and translocation by LplT. Our results suggest that LplT translocates LPLs through an elongated cavity exhibiting an extremely asymmetric polarity. We found that two D(E)N motifs form a head group-binding site, in which the carboxylate group of Asp-30 is important for LPL head group recognition. Substitutions of residues in the head group-binding site disrupted both LPL uptake and flipping activities. However, alteration of hydrophobic residues on the interface between the N- and C-terminal domains impaired LPL flipping specifically, resulting in LPLs accumulation in the membrane, but LPL uptake remained active. These results suggest a dual substrate-accessing mechanism, in which LplT recruits LPLs to its substrate-binding site via two routes, either from its extracellular entry or through a membrane-embedded groove between transmembrane helices, and then moves them toward the inner membrane leaflet. This LPL-flipping mechanism is likely conserved in many bacterial species, and our findings illustrate how LplT adjusts the major facilitator superfamily translocation pathway to perform its versatile lipid homeostatic functions.

Keywords: LplT; homology modeling; lipid flipping; lipid transport; lysophospholipid; major facilitator superfamily; phospholipid turnover; protein-lipid interaction; structural model; structure-function; structure-function study; substrate specificity; transport mechanism; transporter.

Figure 1.

A, thematic representation of the dual-substrate accessing mechanism of LplT in the bacterial inner membrane. LplT recruits LPL substrates (1) from the outer leaflet of the inner membrane generated by Lnt or (2) from the periplasmic space to flip them across the bilayer to the inner leaflet, which are then acylated by Aas to form diacyl lipids on the cytoplasmic surface. B, TLC image of the total phospholipids extracted from spheroplasts generated from E. coli BL21(DE3) ΔlplT cells expressing LplT_Kp WT. C, Western blotting of Lpp in E. coli Trp-3110 WT, ΔlplT, and Δaas, and PAP9502 strains using anti-Lpp antibody. The conditional lnt gene knockout strain PAP9502 was grown in the depleted condition (+glucose) or rescuing condition (+arabinose). The same amount of protein was loaded in each lane. D, [³²P]LPE transport assays of LplT_Kp using spheroplasts prepared from E. coli BL21(DE3) Δaas-lplT strain expressing LplT_Kp WT (black squares) and vector only (open circles) or vector only inside-out vesicles (ISO, open triangles). Radioactivity counts were directly used to calculate the transport activity.

Figure 2.

Docking of lysophospholipids to the LplT_Kp structural models. A, docking scores of 18:1 LPE, LPG, LPA, and LPC to the LplT_Kp WT structural model. B, comparison of substrate docking 18:1 LPE or 18:1 LPG to the LplT_Kp WT, D30N, and K120C mutants. C, docking of LPE, LPG, or LPC with different lengths of the acyl chain (14:1, 16:1, and 18:1) to WT LplT model.

Figure 3.

A, overall architecture of the LplT_Kp structural model in the outward-open conformation with an 18:1 LPE molecule (yellow) docked in the central cavity. The N- and C-domains of LplT_Kp are shown in blue and gray, respectively, whereas the interdomain linker region is shown in red. B, LplT_Kp model viewed from the extracellular side (top), and C, intracellular side (bottom).

Figure 4.

A, distribution of charged residues: basic/positively-charged (blue) and acidic/negatively-charged (red) residues in LplT_Kp model. B, superposition of LplT_Kp model (gray) on YajR_Ec (PDB 3WDO; aquamarine). The conserved motif A in the N-terminal domain of both proteins is highlighted using the dashed square. Other highlighted residues form parts of the C-terminal domain salt-bridge network. Structurally-equivalent residues from LplT_Kp and YajR_Ec are labeled in gray and aquamarine, respectively.

Figure 5.

A–D, sliced section of surface representation showing the orientations of the most favorable docked conformations of (A) 18:1 LPE, (B) 18:1 LPG, (C) 18:1 LPA, and (D) 18:1 LPC within the central cavity of the LplT_Kp model. E, sliced section of the electrostatic potential surface of LplT_Kp calculated by the APBS program (40). Regions having positive (blue) and negative (red) potentials surround the negatively-charged phosphate group and positively-charged ethanolamine head group, respectively, whereas neutral (white) regions surround the acyl tail.

Figure 6.

A, conservation of the cavity-lining residues in the LplT_Kp model. Residues of LplT_Kp were colored according to conservation scores calculated based on 150 sequence homologs (pairwise sequence identity of 35.2–85.6% with LplT_Kp) identified from the UniRef database using the ConSurf web server (41). The 10 residues were identified as having critical roles in LplT_Kp function are shown in sphere representation. B, multiple sequence alignment of 11 LplT sequences from different representative bacterial genera obtained from the UniProt database showing conservation of the 10 critical residues positions (highlighted using same color grades as described for A). The UniProt IDs of the sequences used for producing the MSA are Escherichia (P39196), Shigella (Q32C86), Salmonella (Q8ZMA5), Yersinia (Q1CFA8), Serratia (A8GII5), Enterobacter (A4WE10), Citrobacter (A8AP55), Pectobacterium (C6DE42), Cronobacter (A7MR37), and Photorhabdus (Q7N7A8). C, the binding conformation of 18:1 LPG (blue) in the central cavity. D, 18:1 LPC (magenta) superimposed on the docked conformation of 18:1 LPE (yellow). The residues interacting with the head group are displayed as sticks.

Figure 7.

LPE acylation assays in LplT_Kp spheroplasts. A, TLC images showing conversion of LPE to PE ± 1% Triton X-100 mediated by LplT_Kp WT and mutants. E. coli ΔlplT harboring empty vector served as (−) control. B, the relative LPE acylation activity (% of WT) of mutants. C, Western blotting of LplT_Kp WT and mutant proteins extracted from spheroplasts and developed using anti-His antibody. The same amount of total protein was loaded in each lane.

Figure 8.

A, the conformations of the acyl chain interaction region. The acyl chain of 18:1 LPE is sandwiched between hydrophobic residues on TM2 and Arg-236 from TM7. The residues are depicted as pink sticks. B, V-shaped grooves between TM2 and -11 (orange dashed lines) and between TM5 and -8 (yellow dashed lines), the potential membrane entry. Residue Ile-148 is displayed as a sphere in magenta. C, TLC images of the total phospholipids extracted from spheroplasts generated from E. coli BL21(DE3) ΔlplT expressing LplT_Kp mutants.

Figure 9.

A, polar residues lining the translocation pathway toward the intracellular side of the membrane. 18:1 LPE is depicted in yellow. B, transport activities of LplT_Kp WT and mutants. 4 μm [³²P]LPE or -LPG was added into spheroplasts for 30 min at 37 °C. Error bars represent S.D. of three replicate experiments.