Structural and functional studies of lipopolysaccharide transport and insertion into the outer membrane by LptD/E complex

Antibiotic resistance of bacterial infections is a fast emerging global crisis and an understanding of the underlying resistance mechanisms is paramount for design and development of new therapeutic strategies. Lipopolysaccharides (LPS), which are found in the outer membrane of all Gram-negative bacteria, play a key role in antibiotic resistance. They help to stabilise the lipid membrane and provide a protective barrier that prevents toxic compounds from entering the cell. Although scientists knew which bacterial proteins were involved in the transport to and insertion of LPS into the outer membrane, the mechanism was poorly understood until now.

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In this research, a complex of two transport proteins, LptD and LptE, found in the outer membrane of a class of bacteria called Gram-negative bacteria, was studied. This class of bacteria have a unique outer membrane that is particularly difficult for antibiotics and other drugs to penetrate. To determine the structure of this complex and better understand the mechanism by which this protein transports LPS across the bacterial membrane, the crystal structure of the LptD/E complex was solved using Multi-wavelength Anomalous Dispersion (MAD) data on Macromolecular Crystallography (MX) beamline I24 with the aid of beamline staff. This approach relied on the small beam size and high flux density achievable by I24 to subdivide the crystal and collect 2.86 Å resolution data for each of the 4 wavelengths used. The Pilatus3 detector on I24 facilitated the collection of over 55,000 diffraction images across three crystals in a reasonable time frame that, when combined, allowed the generation of a high quality electron density map and subsequent protein model. The results have identified the path and gate that is used by the bacteria to transport LPS to the outer surface and more importantly that the bacteria would die if the gate was locked shut. These findings provide an exciting platform for future research to develop new compounds that target this specific system; potentially leading to the creation of drugs that bacteria will find it very difficult to become resistant to.

Figure 1: Seven LPS transport proteins form a trans-envelope complex to transport LPS from the inner membrane to the outer membrane.

All Gram-negative bacteria have an outer membrane, which comprises lipopolysaccharide (LPS) in the outer leaflet, and phospholipid in the inner leaflet. LPS is essential for most Gram-negative bacteria and plays a critical role in drug resistance. LPS contains three components: lipid A, core oligosaccharide, and O-antigen. The O-antigen is polymerised by WzY and ligated to the lipid A core oligosaccharide by WaaL1 to form the mature LPS on the exterior face of the inner membrane. Seven LPS transport proteins (LptA-LptG) have been identified as responsible for transport of the mature LPS from the inner membrane to the outer membrane (Fig. 1). Initially the inner membrane associated LptBFG complex uses the energy from ATP hydrolysis to transfer LPS from this membrane to LptC3. LPS is then guided across the bacterial periplasm on a ‘slide’ formed from LptC, LptA, and the N-terminal domain of LptD.

Figure 2: The S. typhimurium LT2 LptD/E complex structure. The LptD forms a 26 β-stranded barrel, while LptE is located inside of the barrel.

At the outset of this research, the mechanism by which the LptD/E complex translocates LPS across the outer membrane and inserts it into the outer leaflet was not understood. As the LptD/E complex is accessible from the exterior of the cell, it was conceivable that drugs targeting this complex may not need to enter the bacterial cell and thus sidestep both the LPS-rich outer membrane and also the efflux pumps that form key bacterial weapons in resisting antibiotics4. The research, aided by results gathered on I24, aimed to determine the structure of the S. typhimurium LT2 LptD/E complex structure and evaluate the novel LPS transport and insertion mechanism. Such structural information would also be invaluable for subsequent antibiotic development.

LptD contains two domains, an N-terminal domain and a C-terminal domain. The N-terminal domain forms a roll structure, which is similar to LptA and LptC. The C-terminal domain forms a 26 β-strand barrel, which is inserted into and spans the outer membrane (Fig. 2). LptE is located inside the LptD barrel as a plug, of which two thirds of LptE are on one side of the barrel with the rest extending into the periplasm. The crystal structure of the LptD/E complex is in the closed form, where 13 extracellular loops and LptE seal the pore of LptD. This maintains the integrity of the outer membrane.
The lumen of LptD barrel is very hydrophilic, and the core oligosaccharide and O-antigen of LPs are translocated across the membrane through the barrel. LptE binds LPS, which helps the transport of LPS through the LptD barrel. LptD and LptE form a stable complex, in which 23 residues of LptE interact with 51 residues of LptD, generating a SDS-stable complex. Mutation of the LptD and LptE interaction residues impairs E. coli cell growth in LB medium containing 0.5% SDS and 1 mM EDTA.
The N-terminal domain of LptD could transport LPS using similar mechanisms to those of LptA and LptC, by locating the lipid A of LPS in the hydrophobic core. By examining the 26 β-stranded barrel, it was clear that β strands 1 and 26 are partially separated on the periplasmic side. It was proposed that the hydrophobic lipid A of LPS may insert into the outer membrane through this gap after the N-terminal domain of LptD delivers LPS to the LptD barrel. A substantial lateral opening would be required to allow the insertion of LPS into the outer leaflet of the outer membrane and such a lateral opening is possible as there are only five hydrogen bonds between β strands 1 and 26 (Fig. 3).

Figure 3: The separation between strands β1 and β26 at the periplasmic side, and possible lateral opening for LPS insertion. (a) the separation between strands β1 and β26 of LptD at the periplasmic side; (b) the functional assays for the double cysteine mutation.

To test this hypothesis, molecular dynamics simulations and mutagenesis studies were performed. The results from the molecular dynamics simulations suggest that the interaction between strands β1 and β26 was the weakest point in the barrel and could separate to allow LPS insertion concurrent to the pore of the barrel opening for core oligosaccharide and O-antigen translocation to the cell surface. Residues N232 and N757 are located in strands β1 and β2 respectively and it was hypothesised that a double cysteine mutation may form a disulphide bond, preventing the lateral separation necessary for LPS insertion into the outer membrane. Cells with the single N232C or N757C mutations of LptD grow similarly to wild type LptD, but the double mutation N232C/N757C kills the bacteria (Fig. 3). This is strong evidence that the lateral opening between strands 1 and 26 is required for LPS insertion into the outer membrane.
In summary, multi-drug resistant Gram-negative bacterial infections are a global health problem, and novel antibiotics are required to control the infection. The LptD/E structural and functional studies provide a new platform for the development of novel antibiotics.

Source publication:
Dong, H., Xiang, Q., Gu, Y., Wang, Z., Paterson, N. G., Stansfeld, P. J., He, C., Zhang, Y., Wang, W. & Dong, C. Structural basis for outer membrane lipopolysaccharide insertion. Nature 511, 52-56, doi:10.1038/nature13464 (2014).

References:
1. Raetz, C. R. H. & Whitfield, C. Lipopolysaccharide endotoxins. Annual Review of Biochemistry 71, 635-700, doi:10.1146/annurev. biochem.71.110601.135414 (2002).

2. Freinkman, E., Chng, S.-S. & Kahne, D. The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel. Proceedings of the National Academy of Sciences of the United States of America 108, 2486-2491, doi:10.1073/pnas.1015617108 (2011).

3. Okuda, S., Freinkman, E. & Kahne, D. Cytoplasmic ATP Hydrolysis Powers Transport of Lipopolysaccharide Across the Periplasm in E-coli. Science 338, 1214-1217, doi:10.1126/science.1228984 (2012).

4. Srinivas, N. et al. Peptidomimetic Antibiotics Target Outer-Membrane Biogenesis in Pseudomonas aeruginosa. Science 327, 1010-1013, doi:10.1126/science.1182749 (2010)

Funding acknowledgement:
Wellcome Trust career development fellowship and Wellcome Trust New Investigator Award to CJD.

Corresponding author:
Professor Changjiang Dong, University of East Anglia, [email protected]

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