Structural mechanisms of pump assembly and drug transport in the AcrAB–TolC efflux system
- State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, China
- Health and Wellness, City University of Macau, China
Figures
Figure 1 with 3 supplements
Structure of the TolC–YbjP complex (closed state).
(A, B) Cryo-EM density map of the endogenous TolC–YbjP complex at 3.56 Å resolution, shown in top and side views, spanning the outer membrane (OM) and periplasmic space. TolC forms a homotrimeric ‘channel-tunnel’ ~14 nm long, with its 12-stranded β-barrel embedded in the OM and periplasmic coiled-coils sealed at the tip. YbjP (wheat) wraps around the equatorial domain of TolC, with continuous density linking YbjP’s N-terminal Cys19 into the OM micelle region. (C, D) Top view (from the outer membrane) and side view of the TolC–YbjP cartoon model, showing three YbjP molecules arranged symmetrically around TolC’s threefold axis. (E, F) Each YbjP straddles the interface between two TolC protomers, occupying a groove on the TolC surface. TolC is shown as surface and cartoon (each protomer in a different green color) and YbjP in wheat cartoon. YbjP’s globular domain inserts between TolC protomers, while an N-terminal linker connects to the OM.
Figure 1—figure supplement 1
Cryo-EM processing workflow for TolC–YbjP complex.
(A) Schematic representation of the multi-step purification protocol employed to isolate the TolC–YbjP and TolC–YbjP–AcrABZ complexes. (B) Cryo-EM processing workflow. (C) Local resolution distribution in final map (Kucukelbir et al., 2014). (D) Gold-standard Fourier Shell Correlation curves for final reconstruction (Rosenthal and Henderson, 2003). (E) Angular distribution plot for final map.
Figure 1—figure supplement 2
Local EM density of YbjP in TolC–YbjP complex.
The region and some bulky residues are labeled. EM density threshold: 8σ.
Figure 1—figure supplement 3
Structural comparison of the anchoring domains from six prototypical efflux pump outer membrane factors.
Cartoon representations of the protomer structures from E. coli TolC–YbjP (PDB: 9V52; this study), K. pneumoniae TOprJ1 (AlphaFold model/PDB: 9J3D) (Shi et al., 2025), C. jejuni CmeC (PDB: 4MT4) (Su et al., 2014), E. coli CusC (PDB: 3PIK) (Kulathila et al., 2011; Su et al., 2011), P. aeruginosa OprM (PDB: 3D5K) (Phan et al., 2010), and V. cholera VceC (AlphaFold model/PDB: 1YC9) (Federici et al., 2005) are shown. The conserved structural region responsible for anchoring the pump to the outer membrane is highlighted within the dashed box for each structure.
Figure 2 with 3 supplements
Architecture of the fully assembled TolC–YbjP–AcrABZ efflux pump.
(A, B) Top and side views of the cryo-EM density map (3.39 Å) of the endogenous TolC–YbjP–AcrABZ complex, with the inner membrane (IM) and outer membrane (OM) boundaries indicated (black bars). The map reveals the complete pump spanning 33 nm. Densities for TolC (green) are supposed to be in the OM region, AcrB (blue) in the IM region as a trimer, and six AcrA protomers (purple) forming an elongated barrel bridging TolC and AcrB. Three AcrZ protomers (pink) are seen at the periphery of the AcrB trimer in the IM region. (C, D) Cartoon models of TolC–YbjP–AcrABZ in top and side views. Three AcrB protomers (blue) form a trimeric base, surrounded by six AcrA molecules (purple) and three AcrZ helices (pink). Each AcrB protomer consists of funnel, porter, and transmembrane domains. (E) Structural alignment of the two AcrA conformations (AcrA and AcrA*) reveals differences in the orientation of the membrane-proximal domain. (F) Zoomed-in view of the TolC–AcrA interface, showing the tight interaction between TolC’s periplasmic helix-turn-helix motifs and AcrA’s α-helical hairpin domain. (G, H) Close-up views of the interface between AcrA (purple), AcrA* (light purple), and AcrB. Panel (G) shows the frontal cartoon representation; panel (H) displays a 60° rotated view, highlighting the intimate packing of AcrA with AcrB.
Figure 2—figure supplement 1
Cryo-EM processing workflow for TolC–YbjP–AcrABZ complex.
(A) Cryo-EM processing workflow. (B) Local resolution distribution in C1 final map (Kucukelbir et al., 2014). (C) Gold-standard Fourier Shell Correlation curves for C1 final reconstruction (Rosenthal and Henderson, 2003). (D) Angular distribution plot for C1 final map. (E) Local resolution distribution in C3 final map (Kucukelbir et al., 2014). (F) Gold-standard Fourier Shell Correlation curves for C3 final reconstruction (Rosenthal and Henderson, 2003). (G) Angular distribution plot for C3 final map.
Figure 2—figure supplement 2
Local EM density of TolC and AcrA in TolC–YbjP–AcrABZ complex.
The region and some bulky residues are labeled. EM density threshold: 8σ.
Figure 2—figure supplement 3
Local EM density of AcrB and AcrZ in TolC–YbjP–AcrABZ complex.
The region and some bulky residues are labeled. EM density threshold: 5σ.
Figure 3
Conformational changes upon pump assembly: closed to open TolC transition.
(A) Side view of TolC–YbjP complex. TolC is closed by coiled-coil helices. (B) Side view of TolC–YbjP part in TolC–YbjP–AcrABZ complex. TolC is in open state. (C) Comparison of TolC in the TolC–YbjP complex (closed state, forest green) and in the TolC–YbjP–AcrABZ pump (open state, pale green). Upon assembly with AcrABZ, these contacts are disrupted and the TolC helices tilt outward, enlarging the aperture. The OM β-barrel domain remains static. YbjP positions are consistent in two structures. (D, E) Top views of TolC–YbjP in closed vs. open states. In the closed conformation, the coiled-coil helices bundle tightly, leaving a ~4 Å diameter sealed pore. In the open state (pale green), the helices are splayed apart, creating a ~20 Å diameter open channel. (F) Quantitative comparison of pore radii in closed and open TolC, as computed using HOLE software (Smart et al., 1996).
Figure 4 with 2 supplements
Conformational cycling of AcrB monomers within the functional trimer.
(A) Structural superimposition of the three AcrB protomers (L, T, and O states) showing their sequential conformational transitions during the transport cycle. (B) Conformational changes in the transmembrane domain associated with proton translocation. (C) Substrate-binding pocket architecture in a single protomer, highlighting subdomains involved in drug recognition. (D) Large-scale structural rearrangements in the porter domain that facilitate substrate translocation.
Figure 4—figure supplement 1
Conformational transitions in AcrB’s porter domain during the transport cycle.
Structural superposition of porter domain (subdomains PN1, PN2, PC1, and PC2) of monomers between L and T states and T and O states, revealing key conformational rearrangements.
Figure 4—figure supplement 2
Structural comparison of AcrB conformational states.
(A) Comparison of the AcrB L state from the holocomplex with known L-state structures (PDB: 3AOB and 3AOC) (Nakashima et al., 2011). Top panels show the funnel and porter domains (top view); bottom panels show a single protomer (side view). (B) Comparison of the AcrB T state with reference T-state structures (PDB: 2DR6, 4DX5, and 5NC5) (Wang et al., 2017; Eicher et al., 2012; Murakami et al., 2006). Panels show top views of the funnel and porter domains (top) and transmembrane domains (middle), and a side view of a protomer (bottom). (C) Detailed comparison of the AcrB O state with 4DX5 (Eicher et al., 2012), using the same three views as in (B). (D) Comparison of the AcrB structure from the holocomplex with an AcrB structure determined in the native membrane environment (PDB: 9DXN). (E) Comparison of the AcrB transmembrane domain with that of the homologous MexB (PDB: 6TA5) (Glavier et al., 2020).
Figure 5
Proposed model of pump assembly and membrane anchoring.
Schematic illustration of the TolC–YbjP–AcrABZ efflux pump within the Gram-negative cell membrane. YbjP is hypothesized to associate with the outer membrane via its N-terminal Cys19, which may undergo lipidation in vivo. YbjP lies just beneath the outer membrane and above the peptidoglycan layer, according to prior electron tomography studies (Shi et al., 2019). In the absence of AcrABZ, TolC adopts a closed state stabilized by inward-pointing periplasmic helices. AcrA, also likely lipid-anchored at its N-terminus, stabilizes the AcrBZ trimer near the inner membrane. Upon engagement with AcrA’s α-helical hairpin domain, TolC helices rotate outward in a left-handed (counterclockwise) manner to open the channel and enable small-molecule substrates to be exported through the fully assembled complex.
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Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/109684/elife-109684-mdarchecklist1-v1.pdf
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Supplementary file 1
Cryo-EM data collection, refinement and validation statistics.
- https://cdn.elifesciences.org/articles/109684/elife-109684-supp1-v1.docx
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Structural mechanisms of pump assembly and drug transport in the AcrAB–TolC efflux system
eLife 14:RP109684.
https://doi.org/10.7554/eLife.109684.3
