Regulatory mechanisms of lipopolysaccharide synthesis in Escherichia coli

Abstract

Lipopolysaccharide (LPS) is an essential glycolipid and forms a protective permeability barrier for most Gram-negative bacteria. In E. coli, LPS levels are under feedback control, achieved by FtsH-mediated degradation of LpxC, which catalyzes the first committed step in LPS synthesis. FtsH is a membrane-bound AAA+ protease, and its protease activity toward LpxC is regulated by essential membrane proteins LapB and YejM. However, the regulatory mechanisms are elusive. We establish an in vitro assay to analyze the kinetics of LpxC degradation and demonstrate that LapB is an adaptor protein that utilizes its transmembrane helix to interact with FtsH and its cytoplasmic domains to recruit LpxC. Our YejM/LapB complex structure reveals that YejM is an anti-adaptor protein, competing with FtsH for LapB to inhibit LpxC degradation. Structural analysis unravels that LapB and LPS have overlapping binding sites in YejM. Thus, LPS levels control formation of the YejM/LapB complex to determine LpxC protein levels.

Fig. 1. LapB is an adaptor specific for FtsH-mediated LpxC degradation.

a Kinetic analysis of LpxC degradation with proteoliposomes (PL). The initial rates of LpxC degraded by FtsH or FtsH/LapB proteoliposomes were fitted to the Michaelis-Menten equation, and K_M and V_max were estimated. b Effect of replacing LapB’s TM helix. His-tagged wild-type or chimeric LapB proteins were expressed and affinity-purified from bacteria, and co-elution of FtsH was examined. The normalized ratio of SDS-PAGE intensities of co-eluted FtsH and LapB bands are listed below the lane of elution. Sup., supernatant of detergent-solubilized membrane after ultracentrifugation; F.T., flow-through. c LapB_cyto concentration-dependent inhibition of LpxC degradation by the FtsH/LapB proteoliposomes. The degradation rate without LapB_cyto was set to 100% and used to normalize rates of LpxC degradation with different LapB_cyto concentrations. For the kinetic analysis and inhibition assay, each experiment was repeated three times and all data were presented as mean values with error bars representing standard deviations (SDs) of triplicates. Source data for (a) and (c) are provided as a Source Data file.

Fig. 2. CryoEM structure of the YejM/LapB complex.

a Surface view of 3D reconstruction of the YejM/LapB complex, filtered to 3.9 Å resolution. Volumes for proteins are presented with a contour level of 0.25. Molecules of LapB_A, LapB_B, YejM_C, and YejM_D are colored in brown, cyan, purple, and pink, respectively. The volume for the detergent micelle is smoothened with a Gaussian filter and shown as a transparent outline. b Ribbon diagram of an atomic model of the YejM/LapB complex, with the thick blue lines indicating the boundaries of the inner membrane (IM). c Side and top views of the TM domains of the YejM/LapB complex, with a pseudo-two-fold axis shown as a dashed line or a dot.

Fig. 3. Lipid-mediated interactions between the YejM_C and the LapB dimer.

a Interactions between the YejM_C and each LapB molecule are located in the different leaflets of the IM. The thick blue lines indicate the boundaries of the inner membrane (IM) and the dashed line indicates the interface of two leaflets. Dashed rectangles highlight protein/protein interactions in each leaflet. Residues involved in interactions within 3.5 Å are shown as balls and sticks. The “empty” spaces at the interfaces are shaded with grey trapezoids. b Lipids at the interface between YejM_C and the LapB dimer. Atomic models of lipid molecules are shown as sticks and balls within their cryoEM densities (green, presented with a contour level of 0.25).

Fig. 4. Structural comparison of the YejM/LapB and YejM/LPS complexes and protease assays with reconstituted proteoliposomes.

a Structural comparison of the YejM_C molecule (purple, residues 210-YPMTARRF-217 in the linker region colored in orange) and the crystal structure of YejM (green, residues 210-YPMTARRF-217 in the linker region colored in dark yellow) (PDB ID: 6XLP), by superimposing the TM domains with an RMSD of 1.22 Å in the main chains. The orientations of the linker in the two structures are highlighted with dashed lines. b Side and top views of the TM domains of the YejM_C and the LapB dimer. c Side and top views of the TM domain of YejM with an LPS molecule (shown as spheres) in the crystal structure. d Protease activity on LpxC of proteoliposomes reconstituted with FtsH, FtsH/LapB, FtsH/LapB/YejM^WT, or FtsH/LapB/YejM^3D (with mutations ofT213D/R215D/R216D). The activity of FtsH-proteoliposomes is set to 1, and the activities of all the other proteoliposomes are normalized to it. Each experiment was repeated three times and all data were presented as mean values with error bars representing standard deviations (SDs) of triplicates. Source data for (d) are provided as a Source Data file.

Fig. 5. Model for regulation of LpxC degradation.

a In exponential growth phase, YejM and LapB form a stable complex, and FtsH degrades LpxC slowly. b In stationary phase, LPS accumulates in the IM, displacing the LapB dimer from YejM. The liberated LapB dimer binds to FtsH, acts as an adaptor protein, and stimulates LpxC degradation.