Lipopolysaccharide is Inserted into the Outer Membrane through An Intramembrane Hole, A Lumen Gate, and the Lateral Opening of LptD

Gu, Yinghong; Stansfeld, Phillip J.; Zeng, Yi C.; Dong, Haohao; Wang, Wenjian; Dong, Changjiang

doi:10.1016/j.str.2015.01.001

Cited by 71 publications

(89 citation statements)

References 43 publications

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“…The 40 C-terminal residues of LptE are apparently disordered and have not been observed in any crystal structure to date. As observed previously (Gu et al, 2015), a lumenal gate postulated to allow core oligosaccharide and O-antigen access to the extracellular surface is formed by two lumenal loops: lumenal loop 1 connects strand β1 of the LptD barrel to its N-terminal domain, while lumenal loop 2 connects strand β26 to the LptD C-terminus. In our truncated structures, lumenal loop 1 is ordered but lumenal loop 2 has no visible electron density despite the presence of the LptD C-terminus in the barrel.…”

Section: Resultssupporting

confidence: 55%

“…The availability of the first high resolution structures of LptDE shed light on the LPS insertion mechanism, including identification of a putative lateral gate through which LPS can be inserted into the outer membrane (Dong et al, 2014; Qiao et al, 2014). Further mutagenic analysis using an LptD depletion system identified regions of the structure that are important for function (Gu et al, 2015) and recently, LPS was shown to crosslink to a number of sites within LptDE, outlining a pathway for LPS transport (Li et al, 2015). …”

Section: Introductionmentioning

confidence: 99%

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Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens

et al. 2016

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SUMMARY Incorporation of lipopolysaccharide (LPS) into the outer membrane of Gram-negative bacteria is essential for viability, and is accomplished by a two-protein complex called LptDE. We solved crystal structures of the core LptDE complexes from Yersinia pestis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and a full-length structure of the K. pneumoniae LptDE complex. Our structures adopt the same plug and 26-strand β-barrel architecture found recently for the Shigella flexneri and Salmonella typhimurium LptDE structures, illustrating a conserved fold across the family. A comparison of the only two full-length structures, SfLptDE and our KpLptDE, reveals a 21° rotation of the LptD N-terminal domain that may impart flexibility on the trans-envelope LptCAD scaffold. Utilizing mutagenesis coupled to an in vivo functional assay and molecular dynamics simulations, we demonstrate the critical role of Pro231 and Pro246 in the function of the LptD lateral gate that allows partitioning of LPS into the outer membrane. eTOC BLURB Crystal structures of the lipopolysaccharide transporter LptDE from three bacterial pathogens reveal new features of the LPS transport mechanism. The N-terminal domain of LptD, which accepts transported LPS from the periplasmic protein LptA, undergoes a large rotation that may facilitate assembly of the LptCAD scaffold.

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Section: Resultssupporting

confidence: 55%

Section: Introductionmentioning

confidence: 99%

Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens

et al. 2016

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“…The two mutations described herein, lptD4213and lptD208, are overlapping deletions in LptD, a ␤-barrel protein responsible for the final step of LPS transport and assembly in the OM (40,41,70,71). Based on recent crystal structures (97,98), both mutations lead to the deletion of the alphahelical loop L4 situated on the extracellular surface of the protein, as well as a portion of either ␤7 or ␤8, respectively. Deletion of this loop may impair LptD function by reducing a few hydrogen bond interactions with the LptE plug and increasing extracellular access to the lumen of the ␤-barrel.…”

Section: Discussionmentioning

confidence: 99%

Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

Balibar

Grabowicz

2016

Antimicrob Agents Chemother

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bGram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens. With emerging multidrug resistance and a limited number of treatment options, bacterial infections pose an ever growing threat to human health. Gram-negative bacteria are particularly recalcitrant to antimicrobial intervention due to their cell envelope structure. Possessing two membranes with different chemical properties (1) and containing an arsenal of efflux pumps (2), Gram-negative bacteria are capable of both excluding and expelling molecules, rendering them resistant to many drugs. Despite major Gram-negative pathogens being classified as urgent or serious threats by the CDC (3), no Gram-negative-active antibiotic with a new mechanism of action has been introduced in over 40 years. Of the 28 new antibiotics approved since 2000, only 18 are indicated for treatment of Gram-negative bacteria (4), and all of those are derived from the four legacy classes ␤-lactams, tetracyclines, macrolides, and fluoroquinolones, which were first introduced in 1942 (5), 1948 (6), 1952 (7), and 1967 (8), respectively. Novel derivatives of old antibiotics often have limited spectra of coverage and can only do so much to overcome existing mechanisms of resistance; therefore, introduction of novel classes of antibiotics is critical.There is no shortage of potential targets in the antibacterial space given that the essential gene set for several Gram-negative pathogens has been defined (9-12). However, despite genetically demonstrating target essentiality, cognate inhibitors with exquisite in vitro activity often have no measurable cellular activity (13,14). The reasons for this can be attributed to a lack of penetration of compounds into bacteria, efflux of molecules out of bacteria, insufficient inhibition of the target, metabolism within the cells, or a combination of the aforementioned factors. Understanding the reason for failure to translate enzymatic 50% inhibitory concentrations (IC 50 s) into cellular MICs is paramou...

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“…Thus, once LPS is delivered to the N-terminal domain of LptD, a conformational change in LptDE would occur, enabling the saccharide portion of the LPS molecules to enter the ␤-barrel and travel to the cell surface, passing through the lateral gate of LptD. On the contrary, it has been proposed that the lipid A domain of LPS would pass first inside the ␤-jellyroll structure of the LptD N-terminal domain and then through the hydrophobic intramembrane open between the N-terminal and the ␤-barrel domains of LptD (66,79,80) (Fig. 2D).…”

Section: Minireview: the Lipopolysaccharide Transport (Lpt) Machinerymentioning

confidence: 99%

The lipopolysaccharide transport (Lpt) machinery: A nonconventional transporter for lipopolysaccharide assembly at the outer membrane of Gram-negative bacteria

Sperandeo

Martorana

Polissi

2017

Journal of Biological Chemistry

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The outer membrane (OM) of Gram-negative is a unique lipid bilayer containing LPS in its outer leaflet. Because of the presence of amphipathic LPS molecules, the OM behaves as an effective permeability barrier that makes Gram-negative bacteria inherently resistant to many antibiotics. This review focuses on LPS biogenesis and discusses recent advances that have contributed to our understanding of how this complex molecule is transported across the cellular envelope and is assembled at the OM outer leaflet. Clearly, this knowledge represents an important platform for the development of novel therapeutic options to manage Gram-negative infections.

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Lipopolysaccharide is Inserted into the Outer Membrane through An Intramembrane Hole, A Lumen Gate, and the Lateral Opening of LptD

Cited by 71 publications

References 43 publications

Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens

Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens

Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

The lipopolysaccharide transport (Lpt) machinery: A nonconventional transporter for lipopolysaccharide assembly at the outer membrane of Gram-negative bacteria

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