LPS transporter (Lpt)
December 2024
The lipopolysaccharide transporter (Lpt) is a multi-protein complex made up of seven proteins (LptA-LptG) that span the inner and outer membranes of Gram-negative bacteria. This complex mediates the transport of lipopolysaccharide (LPS) from inside the bacterial cells to their outer membrane. LPS is a large glycolipid essential for maintaining the integrity of the outer membrane in Gram-negative bacteria, which contributes to their exceptional resistance to antibiotics.
Antibiotic resistance in bacteria poses a global threat to public health, highlighting the need for innovative antibiotic treatments. Earlier this year, a team from Roche identified the Lpt complex as a target for a new class of antibiotics that are currently undergoing clinical trials.
Recently, the same Roche team disclosed the mechanism of action for these novel antibiotics, which was elucidated through high-resolution cryo-EM structural analysis. These molecules block the LPS transport, thus causing an easier disruption of the bacteria's outer membrane. This unprecedented mechanism of inhibition lays the foundation for designing novel, efficacious antibiotics.
Researchers determined the cryo-EM structure of the inner membrane components of the A. baylyi (a Gram-negative bacteria) Lpt complex (LptB2FG) bound to the macrocyclic peptide, compound 1 (Image 1A), a molecule from the new class of antibiotics. They found that compound 1 formed an extensive network of interactions (Image 1B) with both:
The Lpt protein complex, engaging several amino acids in the transmembrane (TM) helices of LptF and LptG. Biochemical studies on mutants of LptFG confirmed that residues on these two proteins are crucial for drug efficacy.
The native ligand LPS, which was always found as a co-complex in all structures. This finding suggests that these drugs are capable of binding LPS within the transporter, trapping an LPS-bound conformation of Lpt.
Image 1. A) Macrocyclic peptide compound 1. At the top , the 3D structure of the compound in the binding site (PDB: 8FRL). At the bottom is the 2D chemical structure of the molecule. B) Cryo-EM high-resolution structure of the A. baylyi Lpt complex (LptB2FG, PDB: 8FRL. LptF is in pink, LptG in grey) bound to LPS (in yellow) and compound 1 (in light blue). The interaction network between the drug, the Lpt residues, and the native ligand LPS are represented as dotted lines, color-coded in light blue for aromatic and yellow for polar contacts.
To better elucidate the mechanisms of inhibition, scientists also solved the structure of LptB2FG bound to LPS in the absence of the drug compound 1 and compared this with the ternary complex including 1 (Image 2). They observed that the overall conformation and contacts of LPS with the transporter protein were nearly identical in both structures. This indicates that compound 1 binds a pre-existing, LPS-loaded state of the transporter, and, therefore, the LptB2FG:LPS complex is the druggable target for the antibiotics.
Image 2. Overlay of the LPS binding site of complex Lpt:LPS:1 (PDB: 8FRL; LptF in pink, LPS in yellow, compound 1 in light blue) to the complex Lpt:LPS (PDB: 8FRM; LptF in blue, LPS in light green). Notice that the LPS conformation in both complexes is almost identical, indicating that the interaction with compound 1 does not alter LPS binding. The drug seems to trap an LPS-bound state of the Lpt complex.
LptC is another member of the inner membrane Lpt complex that was not included in the structures previously discussed. It is known to play an essential role in the transport of LPS from LptF to the outer membrane through a mobile TM helix that can either move away from the transporter or be accommodated between LptG (helix 1) and LptF (helices 5 and 6). When comparing the structure of the Lpt complex without LptC (LptB2FG-LPS-1) to the one with LptC (Image 3), it was observed that compound 1 binds to the region that would typically fit LptC helix, suggesting that the binding of the drug competes with LptC. Any attempts to produce the complete complex with both compund 1 and LptC proved unsuccessful, further supporting this theory. The LptC helix state away from the Lpt complex, likely allows LPS to bind LptFG in the intermediate transport state, creating a pocket that is targeted by compound 1 (and analogs).
Image 3. Comparison of the LPS binding site for the complex Lpt containing the LptC protein (LptB2FGC; PDB: 8FRP; LptFG in light orange, LptC in red) and without LptC (LptB2FG:LPS:1 complex; PDB: 8FRL; LptF in pink, LptG in grey, LPS in yellow, compound 1 in light blue). Notice that the LptC helix occupies the same region of one of compound 1 substituents. This is an indication that the “closed” conformation of the LptC helix would prevent drug binding, and that compound 1.
The insights gained from the structural analyses provided a molecular understanding of the unusual inhibition mechanism and revealed a druggable conformation of the Lpt transporter. This could be further exploited for the structure-based design of innovative antibiotics targeting other Gram-negative pathogens.
Reference:
Pahil, K.S., Gilman, M.S.A., Baidin, V. et al. A new antibiotic traps lipopolysaccharide in its intermembrane transporter. Nature 625, 572–577 (2024). https://doi.org/10.1038/s41586-023-06799-7