Visualizing conformation transitions of the Lipid II flippase MurJ

Abstract

The biosynthesis of many polysaccharides, including bacterial peptidoglycan and eukaryotic N-linked glycans, requires transport of lipid-linked oligosaccharide (LLO) precursors across the membrane by specialized flippases. MurJ is the flippase for the lipid-linked peptidoglycan precursor Lipid II, a key player in bacterial cell wall synthesis, and a target of recently discovered antibacterials. However, the flipping mechanism of LLOs including Lipid II remains poorly understood due to a dearth of structural information. Here we report crystal structures of MurJ captured in inward-closed, inward-open, inward-occluded and outward-facing conformations. Together with mutagenesis studies, we elucidate the conformational transitions in MurJ that mediate lipid flipping, identify the key ion for function, and provide a framework for the development of inhibitors.

Fig. 1

Crystal structures of the Lipid II flippase MurJ_TA. a Chemical structure of the peptidoglycan precursor Lipid II. The third residue of the pentapeptide is variable (shown here is l-lysine but could be meso-diaminopimelate in many Gram-negative bacteria). b Lipid II is synthesized by the combined transferase activities of MraY (PDB 4J72) and MurG (1F0K), then flipped across the cytoplasmic membrane by MurJ for subsequent incorporation into the cell wall. These steps constitute a prototype lipid-linked oligosaccharide (LLO) transport system. c MurJ contains two 6-helix bundles (N-lobe, blue, and C-lobe, green) common to the MOP superfamily and 2 additional C-terminal helices (brown). d N-lobe and C-lobe are related by pseudo-twofold rotational symmetry normal to the membrane (top view is shown). Symmetry is distorted at TMs 1, 2, 7, and 8 which enclose the central cavity. TMs 13 and 14 form a hydrophobic groove that enters the central cavity through a portal. e Our previous crystal structure of MurJ (5T77), and crystal structures in the inward-closed, inward-open, inward-occluded, and outward conformations (front and bottom views). Resolutions (from left) are 2.0, 3.2, 3.0, 2.6, and 1.8 Å

Fig. 2

Lateral gating mechanism in the inward-facing state. a Superposition of inward-facing structures highlights similarity at the periplasmic gate but divergence at the cytoplasmic gate. b The periplasmic gate is stabilized by hydrogen bonds between Gly245/Ser248 and the essential Asp39. c At the cytoplasmic side, coordinated rearrangement of TM 1 (blue) and TM 8 (green) controls portal dilation. The Cα-Cα distance between Ser11 (TM 1) and Ser267 (TM 8) increases from 8.0 Å in the inward-closed structure to 17.4 Å in the inward-open structure. d Rearrangement of the TM 4–5 loop (orange) and unwinding of the cytoplasmic ends of TMs 4/5 could induce bending of TM 1. The conserved Phe151 (Phe157 in MurJ_EC) provides leverage to bend TM 1 (residues 1–20) out into the membrane. TM 4–5 loop is shown in sausage representation, with thickness proportional to conservation. Sidechains of Arg18 and Phe151 were not resolved in the inward-occluded structure

Fig. 3

The asymmetric inward-occluded conformation of MurJ_TA. a Rotation of the cytoplasmic half of C-lobe by ~15° from the inward-open structure (left) to the inward-occluded structure (right). Conserved residues Pro260, Pro300, and Gly340 serve as hinges. At the same time, the middle segment of TM 2 also bends inwards by ~15°. Together these motions bring Glu57 (TM 2) and Arg352 (TM 10, Lys368 in E. coli MurJ) into proximity, which form a thin gate. Most of TM 1 is hidden for clarity. b The conserved (G/A)-E-G-A motif (orange) allows S-shaped bending of TM 2 by breaking the helix. TM 8 assumes a more α-helical geometry (albeit still not ideal) in the inward-occluded structure than in the other inward structures. c Unmodeled 2Fo − Fc electron density at the portal and transport cavity of the inward-occluded structure, displayed here as purple mesh contoured to 0.8 σ

Fig. 4

The outward conformation of MurJ_TA. a The outward-facing cavity is more shallow and narrow than the inward-facing cavity. b Straightening of TM 7 and concomitant lowering of the TM 6–7 loop could close the cytoplasmic gate, allowing transition from the inward-occluded to the outward-facing conformation. c Alignment of N-lobes and C-lobes from the inward-occluded conformation (gray) to those of the outward-facing conformation (in color), showing rotation of not just TM 7 but also TM 1, the latter closing the lateral membrane portal. d The cytoplasmic gate is mainly stabilized by a hydrogen bond network between TMs 2 and 10, with less-conserved electrostatic interactions at the cytoplasmic loops

Fig. 5

The sodium binding site in MurJ_TA. a Na⁺ is bound in the C-lobe (TMs 7, 11, 12) coordinated by Asp235, Asn374, Asp378, Val390 (backbone carbonyl), and Thr394. Fo − Fc omit density for Na⁺ is shown as magenta mesh contoured to 4.5 σ (peak height ~19 σ, 1.8 Å resolution). 2Fo − Fc omit density is shown in gray mesh. Na⁺ is coordinated by trigonal bipyramidal geometry. b Na⁺ was also bound in the inward-occluded and inward-closed structures. c Na⁺-associated rearrangement could be propagated down TM 7. d Complementation assay of MurJ_TA sodium site mutants in MurJ_EC-depletion strain NR1154. Cells transformed with plasmids encoding MurJ_TA (wild-type or sodium site mutant) or without insert (pEXT21) were depleted of endogenous MurJ by serial dilution on agar plates containing the anti-inducer d-fucose. MurJ_TA expression was induced by addition of IPTG. Data shown are representative of three biological replicates. Source data are provided as a Source Data File. Mutants D235A and N374A were previously determined to neither express nor complement

Fig. 6

State-dependent conformational changes in the central cavity during the transport cycle. a The inward-facing cavity opens up in the inward-open conformation, and then extends deepest into the membrane in the inward-occluded conformation. In contrast, the outward-facing cavity is narrow and shallow. b Systematic rearrangement of the Arg24-Asp25-Arg255 triad in the cavity during the transport cycle. In the inward-occluded state, Arg24 and Arg255 are brought into unusual proximity (just 2.9 Å apart at the closest), with the unfavorable repulsion stabilized by electrostatic interactions with Asp25 and the diphosphate moiety of Lipid II. The lipid-diphosphate model was docked into final electron density (blue mesh, contoured to 1.0 σ) and was never used for refinement or map calculation. c Proposed model of the MurJ transport mechanism