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Structural basis of periplasmic bridge protein docking and nucleotide-dependent remodelling in lipopolysaccharide transport

This study utilizes cryo-electron microscopy, molecular dynamics, and native mass spectrometry to reveal the structural architecture of the *Pseudomonas aeruginosa* LptB2FGC-LptH complex and elucidate how ATP binding drives nucleotide-dependent remodeling of the periplasmic bridge while maintaining its continuity for lipopolysaccharide transport.

Original authors: Jani Bolla, Yi Wang, Matteo Cervoni, Hayden Tucker, Keyi Wei, Corinne Lutomski, Tarick El-Baba, Francesco Imperi, Phillip Stansfeld, Carol Robinson, Francesco Fiorentino, Chih-Chia Su

Published 2026-10-15

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CC BY 4.0

Original authors: Jani Bolla, Yi Wang, Matteo Cervoni, Hayden Tucker, Keyi Wei, Corinne Lutomski, Tarick El-Baba, Francesco Imperi, Phillip Stansfeld, Carol Robinson, Francesco Fiorentino, Chih-Chia Su

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

The outer shell of many harmful bacteria is a fortress built from a unique material called lipopolysaccharide. This substance forms the outer layer of their cell membrane, acting as a shield that blocks antibiotics and keeps the cell intact. To build and maintain this shield, the bacteria rely on a complex assembly line that stretches from the inside of the cell to the outside. This machine pulls the building blocks from the inner membrane, carries them across the empty space in the middle of the cell, and deposits them on the surface. If this transport line breaks, the bacteria cannot survive. For years, scientists have understood the individual parts of this machine, but they have struggled to see how the moving parts connect to the stationary ones, or how the machine knows when to grab a building block and when to let it go.

A team of researchers has now captured a detailed, three-dimensional view of this connection in action. They focused on a specific type of bacteria known as Pseudomonas aeruginosa, which is a common cause of difficult-to-treat infections. Using a powerful imaging technique that freezes molecules in time, the scientists built a model of the transport machine with its bridge component attached. They examined the machine in two different states: first, when it was idle and empty, and second, when it was bound to a molecule that mimics the energy fuel the bacteria uses to power the process. Their work reveals exactly how the bridge locks onto the machine and how the machine reshapes itself to prepare for pushing the cargo forward without breaking the connection.

The researchers found that the bridge, a long protein chain, attaches to the main transporter by snapping together like two pieces of a puzzle. Specifically, the end of the bridge protein fits perfectly against the end of a protein on the machine, extending a continuous, twisted groove that runs from the inner membrane out into the cell. This groove is lined with a water-repelling surface that allows the greasy building blocks of the shield to slide through without touching the watery environment inside the cell. The team confirmed that this connection is stable and forms even before the cargo arrives, suggesting the bridge is assembled and ready to receive its load at any moment.

When the researchers added the energy-mimicking molecule to the mix, the machine changed shape in a very specific way. The two halves of the transporter's engine closed together, squeezing the central cavity where the cargo sits. While the captured structure was empty of cargo, this closing action is what drives the movement of the cargo out of the machine and into the bridge in a functioning system. Crucially, the bridge itself did not fall apart or detach. Instead, the connection point where the bridge meets the machine shifted slightly, acting like a flexible joint that allows the machine to push while the bridge stays attached. This remodeling happens right at the entrance to the bridge, effectively gating the flow of material so it moves in only one direction.

To understand how this system might be attacked, the team tested a natural antimicrobial peptide called thanatin, which is known to disrupt bacterial transport. They observed that this peptide first breaks apart the links between bridge proteins that are not yet attached to the machine. Only at higher concentrations did it begin to disturb the strong connection between the bridge and the transporter. This suggests that the weakest link in the chain is the self-assembly of the bridge proteins, and that the connection to the main machine is much more robust. Computer simulations supported these findings, showing that while the energy-driven closure of the machine makes the bridge connection slightly looser, it remains intact enough to keep the transport line open.

These findings provide a clear picture of how a bacterial transport machine operates as a coordinated unit. The bridge is not just a passive tube; it is a dynamic part of the machine that remains attached even as the engine cycles through its power strokes. The study shows that the bacteria do not need to rebuild the bridge every time they move a piece of cargo. Instead, they maintain a continuous, assembled pathway and use energy to remodel the entrance, ensuring that the greasy cargo is pushed forward efficiently. This detailed understanding of the machine's architecture and its weak points could help scientists design new drugs that specifically jam this transport line, leaving the bacteria vulnerable to attack.

Technical Summary: Structural Basis of Periplasmic Bridge Protein Docking and Nucleotide-Dependent Remodelling in Lipopolysaccharide Transport

Problem Statement
The outer membrane of Gram-negative bacteria relies on the lipopolysaccharide (LPS) transport (Lpt) machinery to extract LPS from the inner membrane and translocate it across the periplasm to the outer membrane. While the individual components of this system—the inner-membrane ABC transporter LptB2FGC, the periplasmic bridge protein LptA (LptH in Pseudomonas aeruginosa), and the outer-membrane translocon LptDE—are known, the structural mechanism governing the engagement between the transporter and the soluble bridge remains unresolved. Specifically, it is unclear how the inner-membrane transporter recruits the bridge component, how the junction responds to the ATPase cycle, and whether nucleotide binding triggers the assembly or merely remodels an already formed complex. Furthermore, the molecular basis for the disruption of these interfaces by antimicrobial peptides like thanatin is not fully defined.

Methodology
The authors employed an integrative structural biology approach combining native mass spectrometry (MS), cryo-electron microscopy (cryo-EM), conditional complementation assays, and molecular dynamics (MD) simulations.

Sample Preparation: P. aeruginosa LptH was expressed and purified, while LptB2FGC was expressed in ClearColi cells producing lipid IVA and subsequently delipidated. The complexes were reconstituted into lipid nanodiscs.
Native MS: Used to assess the stoichiometry and stability of the LptB2FGC-LptH complex in solution and to monitor the sequential disruption of interfaces by the antimicrobial peptide thanatin.
Cryo-EM: Single-particle analysis was performed on the LptB2FGC-LptH complex in two states: nucleotide-free (apo) and AMP-PNP-bound (nucleotide-engaged). Structures were resolved at 3.24 Å and 3.05 Å, respectively.
MD Simulations: 500 ns simulations were conducted on both apo and ATP-bound complexes to analyze dynamic behavior, interface stability, and orientational ensembles in a membrane environment.
Functional Assays: Conditional complementation assays in P. aeruginosa were used to validate the functional importance of specific residues at the LptC-LptH interface.

Key Results

Stable Complex Formation: Native MS confirmed that LptH forms a stable complex with LptB2FGC in the absence of LPS or nucleotides. The transporter can bind up to two molecules of LptH, with the monomeric form being the predominant species in the complex.
Structural Architecture of the Junction: The cryo-EM structures reveal that LptH docks onto the distal end of LptC via edge-to-edge pairing of the C-terminal $\beta$ -strand of LptC with the N-terminal $\beta$ -strand of LptH. This $\beta$ -strand augmentation extends the $\beta$ -jellyroll domains into a continuous, highly twisted antiparallel $\beta$ -sheet, creating a hydrophobic groove that shields LPS from the aqueous periplasm.
Interface Specificity: The LptC-LptH interface is stabilized by a compact hydrophobic core and a network of polar/charged interactions. While individual residues show limited sequence conservation across species, the structural complementarity of the $\beta$ -strands is preserved. Mutational analysis confirmed that disrupting these specific residues abolishes cell viability, validating the interface's functional necessity.
Thanatin Disruption Hierarchy: Native MS and MD simulations demonstrated that the antimicrobial peptide thanatin disrupts Lpt assemblies in a defined sequence. It preferentially targets and destabilizes the LptH-LptH dimeric interaction (which buries less surface area) before disrupting the more extensive and stable LptC-LptH junction at higher concentrations.
Nucleotide-Dependent Remodelling: Upon binding the non-hydrolysable ATP analogue AMP-PNP, the LptB dimer closes, and the transmembrane cavity collapses. Crucially, the soluble LptH bridge remains attached. However, the nucleotide binding induces a conformational change concentrated at the LptF-LptC junction, causing a separation of the $\beta$ -jellyroll domains and narrowing the connection to the membrane cavity. The distal LptC-LptH segment remains assembled but becomes slightly less compact and more flexible, as shown by MD simulations.

Significance and Claims
The paper establishes the atomic architecture of the transporter-bridge junction in P. aeruginosa, defining how LptH docks onto LptC to complete the periplasmic conduit. The authors claim that bridge assembly is a pre-requisite step that can occur independently of LPS loading or nucleotide binding.

A central finding is that nucleotide engagement does not trigger the recruitment or dissociation of the bridge component; rather, it remodels an already assembled complex. The study reveals an asymmetric structural response where ATP binding drives the closure of the inner-membrane transporter and selectively remodels the entrance to the periplasmic bridge (at the LptF-LptC junction) while preserving the continuity of the distal bridge (LptC-LptH). This supports a model where the Lpt system operates as a coupled machine, with nucleotide-driven closure acting as a checkpoint that controls access to the periplasmic conduit without breaking the bridge.

Finally, the work elucidates the mechanism of action for the antimicrobial peptide thanatin, showing it exploits the relative weakness of LptH self-association compared to the transporter-proximal interface. These findings provide a structural basis for understanding the coordinated dynamics of LPS transport and offer insights into potential pharmacological strategies targeting protein-protein interactions within the Lpt pathway.

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