Structural mechanisms of pump assembly and drug transport in the AcrAB-TolC efflux system

This study presents high-resolution cryo-EM structures of native *E. coli* efflux complexes that reveal the previously uncharacterized lipoprotein YbjP acts as a crucial structural component anchoring TolC to the outer membrane and accommodating conformational changes during the complete drug transport cycle.

The Gist

The Big Picture: Bacteria's "Super-Drain"

Imagine a Gram-negative bacterium (like E. coli) as a high-security fortress with two layers of walls: an inner wall and an outer wall. Inside this fortress, the bacteria are constantly under attack by antibiotics, which are like enemy missiles trying to get in and destroy the bacteria.

To survive, these bacteria have built a three-part "super-drain" system (called the AcrAB-TolC pump). This system acts like a giant vacuum cleaner that sucks up the enemy missiles (antibiotics) from inside the fortress and blasts them out through the outer wall before they can do any damage. This is why bacteria become "superbugs" and resist our medicines.

For a long time, scientists knew how the vacuum cleaner worked, but they didn't fully understand how the outer pipe (the exit tunnel) stayed attached to the wall or how it opened up to let the trash out.

The New Discovery: The "Missing Link" (YbjP)

In this study, researchers used a super-powerful microscope (Cryo-EM) to take 3D pictures of this pump in action. They discovered a previously unknown piece of the puzzle: a small protein called YbjP.

Here is the analogy:

• The Pump: Imagine a long, flexible garden hose that needs to connect a water pump (inside the house) to a drain in the street (outside).
• The Problem: The hose (TolC) doesn't have a built-in hook to stick it to the street curb. Without a hook, it might flop around or fall off.
• The Solution (YbjP): The researchers found that the bacteria uses a special "clamping strap" (YbjP) to tie the hose securely to the curb. This strap wraps around the middle of the hose, holding it tight so it doesn't move, even when the pump is working hard.

How the Pump Works (The Dance of the Three Parts)

The pump is made of three main teams working together:

1. The Inner Team (AcrB): This is the engine. It sits on the inner wall and grabs the antibiotics. It works like a rotating carousel. It has three seats, and each seat goes through three stages:

   • Loose (L): Grabs the antibiotic.
   • Tight (T): Squeezes it tight.
   • Open (O): Shoots it out.
   • Analogy: Think of a trash compactor. One arm grabs the trash, the next arm crushes it, and the third arm kicks it out the back. They do this in a circle, so the machine never stops.

2. The Bridge Team (AcrA): These are the connectors. They are like flexible arms that reach from the inner engine to the outer pipe, passing the message: "Hey, we have trash! Open the door!"

3. The Outer Pipe (TolC): This is the exit tunnel through the outer wall.

   • Closed State: Normally, the tunnel is sealed shut like a closed iris (like a camera lens) so nothing gets in or out.
   • Open State: When the inner engine pushes the bridge team, the outer pipe unfurls. The "iris" opens up, creating a wide tunnel for the antibiotics to fly out.

The "Magic" of YbjP

The most exciting part of this paper is what they learned about YbjP (the clamp):

• It's a permanent anchor: Unlike other bacteria that use a built-in "glue" (lipid) to stick their pipes to the wall, E. coli uses YbjP as a separate helper. YbjP is like a safety harness that wraps around the pipe and hooks it to the wall.
• It's flexible: The researchers saw that even when the pipe opens up (unfurls) to let drugs out, YbjP stays attached. It stretches and moves with the pipe, acting like a shock absorber. It ensures the pipe doesn't rip off the wall while it's doing its job.

Why This Matters

Think of this discovery like finding the missing instruction manual for a complex machine.

• Before: We knew the machine had a pump and a pipe, but we didn't know how the pipe stayed attached or how it opened so smoothly.
• Now: We know that YbjP is the crucial "glue" that holds the system together.

The Takeaway:
By understanding exactly how this "super-drain" is built and how it stays attached, scientists can now look for ways to break the YbjP clamp. If we can make the clamp fall off, the whole pump falls apart, and the antibiotics can finally get in and kill the bacteria. This opens up a new path to fighting superbugs!

Technical Summary

1. Problem Statement

Tripartite multidrug efflux pumps, such as the AcrAB–TolC system in Escherichia coli, are critical determinants of antibiotic resistance in Gram-negative bacteria. These pumps span the entire cell envelope, expelling toxic compounds from the cytoplasm to the extracellular space. Despite extensive study, fundamental gaps remain in understanding:

• TolC Anchoring: Unlike homologous channels in other bacteria (e.g., Pseudomonas OprM or E. coli CusC) that possess intrinsic N-terminal lipid anchors, E. coli TolC lacks a covalent lipid modification. The mechanism by which TolC is stably positioned and retained in the outer membrane (OM) during pump assembly was unknown.
• Structural Resolution: Previous structural studies of the assembled pump were limited by low-to-medium resolution and conformational heterogeneity, preventing accurate modeling of flexible regions and potentially masking the presence of accessory subunits.
• Assembly Mechanism: The precise structural pathway of how the inner membrane transporter (AcrB), the periplasmic adaptor (AcrA), and the outer membrane channel (TolC) assemble into a functional complex was not fully resolved at high resolution.

2. Methodology

The authors employed single-particle cryo-electron microscopy (cryo-EM) to determine high-resolution structures of endogenous efflux complexes purified directly from E. coli membranes.

• Sample Preparation: E. coli C600 cells were cultured, stressed via $\lambda$ phage infection, and lysed. Membrane proteins were extracted using the detergent n-dodecyl-β-D-maltoside (DDM) and purified via size-exclusion chromatography (SEC). This approach preserved native protein-protein interactions without artificial crosslinking.
• Data Collection: Cryo-EM data were collected on a 300 kV Titan Krios microscope equipped with a Gatan K3 Summit detector. Approximately 3,452 movies were acquired.
• Image Processing:
  • TolC–YbjP Subcomplex: Particles were processed using C3 symmetry, yielding a map at 3.56 Å resolution.
  • Full TolC–YbjP–AcrABZ Complex: A larger dataset was processed. Due to the intrinsic asymmetry of the AcrB trimer (which cycles through three states), symmetry expansion and 3D classification were used. Final maps were generated at 3.39 Å (C1 symmetry for the full complex) and 3.26 Å (C3 symmetry for local refinement of symmetric regions).
• Model Building: Atomic models were built using de novo tracing for unknown densities and rigid-body fitting for known components. Protein identity for unknown densities was confirmed using CryoNet and AlphaFold Database searches, followed by manual adjustment in Coot and refinement in PHENIX/Refmac.

3. Key Contributions and Results

A. Discovery of YbjP as a Novel Structural Component

• Identification: The study identified a previously uncharacterized lipoprotein, YbjP (UniProt P75818), bound to TolC.
• Stoichiometry: YbjP binds to the TolC trimer in a 3:3 stoichiometry, with each YbjP molecule bridging two adjacent TolC protomers at their equatorial domain.
• Membrane Anchoring: The structure reveals clear density for the mature YbjN-terminal Cys19, confirming it is anchored to the outer membrane via an N-terminal lipid moiety (predicted to be dually lipidated). This provides the missing "lipid anchor" for TolC, compensating for TolC's lack of intrinsic lipidation.
• Structural Role: YbjP acts as a molecular clamp, stabilizing the closed conformation of TolC and facilitating its proper orientation in the outer membrane.

B. High-Resolution Structure of the Full Pump (TolC–YbjP–AcrABZ)

• Architecture: The complete complex spans ~33 nm, comprising the TolC channel (14 nm), the periplasmic AcrA/AcrB region (14 nm), and the inner membrane AcrBZ (5 nm).
• Stoichiometry: The complex follows a 3:6:3 ratio (TolC:AcrA:AcrB), with three AcrZ molecules bound to the AcrB trimer.
• AcrA Hexamer: Six AcrA molecules form an elongated hexameric ring bridging TolC and AcrB. The structure reveals functional asymmetry between adjacent AcrA protomers (AcrA vs. AcrA*), driven by differential interactions with the TolC equatorial domain and the AcrB funnel domain.
• AcrZ Role: The small transmembrane protein AcrZ (49 aa) is resolved bound to each AcrB protomer, stabilizing the transmembrane helices and potentially modulating conformational dynamics.

C. Mechanism of TolC Gating (Closed-to-Open Transition)

• Iris-like Dilation: Comparison of the closed (TolC–YbjP) and open (TolC–YbjP–AcrABZ) states reveals an iris-like mechanism. The periplasmic coiled-coil helices of TolC undergo a 27° rigid-body superhelical rotation around the equatorial domain.
• YbjP Accommodation: Crucially, YbjP remains bound to TolC in both the closed and open states. This demonstrates that YbjP is flexible enough to accommodate the massive conformational changes required for channel opening without dissociating, acting as a stable scaffold.
• Pore Expansion: The pore diameter expands from ~4 Å (sealed) in the closed state to ~20 Å in the open state, allowing substrate passage.

D. Substrate Transport Mechanism in AcrB

• Rotary Mechanism: The AcrB trimer captures all three distinct conformational states (Loos, Tight, Open) simultaneously within the intact complex.
• Proton Relay: The transition is driven by the proton motive force (PMF). Key residues (D407, D408, K940, R971) in the transmembrane domain facilitate proton translocation.
• Conformational Cycling:
  • L to T: Initiated by a random-coil-to-helix transition in TM8 and an 8 Å shift of the PC2 subdomain, moving substrates from the access pocket (AP) to the deep binding pocket (DBP).
  • T to O: Triggered by protonation of D407/D408, causing inward movement of transmembrane repeats and closing the DBP while opening a tunnel to the funnel domain for extrusion.
• Native Context Differences: The study notes subtle but functionally relevant conformational differences in the AcrB states when observed in the intact endogenous complex compared to isolated AcrB structures, highlighting the influence of native constraints (AcrA and AcrZ binding).

4. Significance

• Resolution of the "Anchoring Paradox": The study solves the long-standing question of how E. coli TolC is anchored to the outer membrane, identifying YbjP as the essential lipidated partner that replaces the intrinsic lipid anchors found in other bacterial homologs.
• Comprehensive Assembly Model: It provides the first high-resolution view of the fully assembled, endogenous tripartite pump, revealing the precise 3:6:3 architecture and the role of the accessory protein AcrZ.
• Mechanistic Insight: By capturing the full transport cycle (L-T-O states) and the TolC gating mechanism in a single high-resolution dataset, the paper offers a unified model of how proton motive force is converted into mechanical work for drug extrusion.
• Therapeutic Implications: Understanding the specific structural interfaces (e.g., YbjP-TolC, AcrA-TolC) and the assembly pathway provides new potential targets for developing adjuvants that could disrupt pump assembly or function, thereby restoring antibiotic efficacy against resistant Gram-negative bacteria.

In summary, Ge et al. utilize advanced cryo-EM to redefine the architecture of the AcrAB–TolC pump, identifying YbjP as a critical structural component and providing a detailed mechanistic view of drug transport and channel gating in its native state.