One-step drug transport across two membranes of Gram-negative bacteria

doi:10.21203/rs.3.rs-4468934/v1

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One-step drug transport across two membranes of Gram-negative bacteria

https://doi.org/10.21203/rs.3.rs-4468934/v1

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Transport of proteins and small molecules across the complex cell envelope of Gram-negative bacteria is crucial for their survival and interaction with their environment and is facilitated by specialized macromolecular machines that enable direct one-step or indirect two-step translocation of substrates. Major facilitator superfamily (MFS)-type tripartite efflux pumps and type I secretion systems likely employ a similar one-step mechanism for substrate transport across cell membranes, but the structural details remain elusive. A representative MFS-type tripartite efflux pump, EmrAB-TolC, mediates multidrug resistance through proton-coupled EmrB, a member of the DHA2 transporter family. Here, we demonstrate that the EmrAB-TolC pump confers resistance to clinical antibiotics, including polymyxin B and neomycin, and report the high-resolution (3.11 Å) structure of the pump, revealing a unique, asymmetric architecture emerging from the TolC:EmrA:EmrB ratio of 3:6:1. This structure identifies two critical subdomains, AssA and AssB, essential for pump assembly and key residues involved in pump assembly, drug recognition, proton translocation and coupling, which are corroborated by mutagenesis and antibiotic sensitivity assays. The delineation of the complete translocation pathway reveals the molecular mechanism for one-step drug transport process across the entire cell envelope.

Biological sciences/Biochemistry/Structural biology/Electron microscopy/Cryoelectron microscopy

Biological sciences/Microbiology/Antimicrobials/Antibiotics

Transport of proteins and small molecules across the cell envelope is essential for the survival of bacteria and interactions with their microbiological niches. The complex architecture of the cell envelope in Gram-negative bacteria presents a considerable obstacle for the translocation of substrates across it. To overcome this barrier, these organisms have evolved a diverse array of specialized macromolecular nanomachines capable of secreting a variety of substrates^1,2. The process of transporting substrates across the outer membrane (OM) can occur through two mechanisms: either the substrates are transported directly from the cytosol in a one-step manner, or alternatively they are transported in a two-step fashion in which substrates are first moved to the periplasm through one transporter system on the inner membrane (IM), and then translocated across the OM by another ². Tripartite efflux pumps and the closely related type I secretion systems (T1SSs) in Gram-negative bacteria are double membrane-spanning nanomachines ^1,2. Three classes of tripartite efflux pumps have been identified: MFS types such as EmrAB-TolC^3,4; RND types exemplified by AcrAB-TolC ^5,6; and ABC types represented by MacAB-TolC ⁷. The AcrAB-TolC and MacAB-TolC pumps operate on a two-step transport mechanism ⁸. The MFS-type tripartite efflux pumps and T1SSs, however, are believed to employ a similar one-step transport mechanism, the structural details of this process remain unclear ^3,4.

The MFS-type tripartite efflux pumps are present in many Gram-negative bacterial pathogens ^3,4. The prototype MFS-type EmrAB-TolC pump of Escherichia coli plays an essential role in mediating multidrug resistance and contributes to virulence phenotypes ^9–13. This pump consists of three components: a TolC OM channel, an EmrB IM transporter, and an EmrA periplasmic adaptor which bridges these two proteins ⁹. Understanding of the overall structure and organization of the EmrAB-TolC pump is currently limited to the structural data of individual, separate components of its homologues ^3,4. The Aquifex aeolicus EmrA structure exhibits a linear arrangement of α-helical hairpin, lipoyl, and β-barrel domains. A conserved loop in the β-barrel domain is much longer than that of other adaptors, which is disordered in the determined structure ¹⁴. EmrB belongs to the DHA2 transporter family. Unlike the well-characterized DHA1 family, for which structures in various conformations are available ^15–18, the DHA2 family has been less well characterized. However, recent studies on an analogous DHA2 transporter QacA have revealed critical acidic residues essential for substrate recognition and transport ¹⁹. Structural analysis of another DHA2 transporter MHAS2168 from Mycobacterium hassiacum revealed the extension of two TM helices, TM 11 and TM12, into the periplasm, which might interact with a lipoprotein for substrate translocation ²⁰. Finally, the stoichiometry of the EmrAB-TolC assembly remains undefined, with studies reporting conflicting models of either 3:6:1 or 3:6:2 TolC:EmrA:EmrB ratios ^14,21,22. The high-resolution structure of the full pump assembly and the mechanism of the pump are currently unknown.

In this study, we present the cryo-EM structure of the E. coli EmrAB-TolC assembly for the first-time, revealing how the pump confers multidrug resistance to clinically important antibiotics. The pump structure identifies key residues that contribute to such multidrug resistance and are corroborated by mutational analysis. The architecture of the EmrAB-TolC pump uncovers a one-step mechanism that directly transport drugs across the entire cell envelope.

Engineering stable and functional EmrAB-TolC complexes

Initial attempts to generate a native EmrAB-TolC pump failed to yield the entire pump assembly, as the protein complex was highly susceptible to both dissociation and precipitation. To stabilize the pump assembly, we first modified EmrB by fusing a thermostabilized apocytochrome b562RIL (BRIL) protein and an ALFA tag to the N- and C-termini of the protein ^23,24, respectively. We then engineered an EmrB-EmrA fusion protein with a flexible linker that connected the modified EmrB and EmrA. We observed that the N-terminus of EmrA and the C-terminus of EmrB are likely adjacent to the cytoplasmic side, and EmrA possesses a single N-terminal TM helix. Consequently, EmrA was fused to the C-terminus of modified EmrB via a flexible polyglycine-serine linker, thereby maintaining the correct membrane topology of the pump components.

Co-expression of the fused EmrB-EmrA protein, free EmrA, and TolC led to the formation of a complex (engineered pump-FA). However, despite successful expression, the complex tended to dissociate or precipitate during purification, resulting in protein concentrations of only ~0.1 mg/mL. Therefore, the N-terminal segment (residues 1-47) of free EmrA (containing the N-terminal TM helix) were replaced by the AcrA signal sequence (residues 1-27) containing residue C25, which can be modified by a palmitoyl acid chain to anchor EmrA to the IM ^25,26. The modified free EmrA, EmrB-EmrA fusion protein and TolC were successfully expressed and copurified, and the complex (engineered pump-EA) exhibited modest stability during purification, achieving a concentration of ~0.7 mg/ml.

Survival assays revealed that the wild-type EmrAB-TolC pump conferred drug resistance to polymyxin B, neomycin, nitroxoline, and nalidixic acid (Extended Data Fig. 1a). Growth assays of E. coli expressing the engineered pumps showed that they conferred resistance to nitroxoline, indicating that the complexes were functional in vivo (Extended Data Fig. 1b).

Quaternary structure of the EmrAB-TolC pump

The structure of EmrAB-TolC was solved using single-particle cryo-EM. Three density maps were reconstituted: two maps for pump-EA achieved overall resolutions of 3.11 Å and 3.14 Å, respectively; and one map for pump-FA reached 3.59 Å resolution (Extended Data Fig. 2a-c; Extended Data Fig. 3a-c). Local resolution examination of these maps indicated a resolution ranging between 3.0 Å to 7.0 Å (Extended Data Fig. 2d; Extended Data Fig. 3d). The map for pump-EA was of sufficient quality to facilitate the building of models for residues 47-390 of EmrA and residues 13-498 of EmrB (Fig. 1; Extended Data Fig. 2e). The map for pump-FA was used to construct models for the extra residues 16-46 of EmrA (Fig. 2a-c; Extended Data Fig. 4).

The pump has an elongated shape consisting of a TolC trimer, an EmrA hexamer and an EmrB monomer (Fig. 1; Extended Data Fig. 4). In the case of pump-FA, the model of the N-terminal TM helix of the EmrA protomer 4 spans the entire IM, while the partial models of the other five EmrA protomers are shorter (Extended Data Fig. 4; Extended Data Fig. 5).

The positions of EmrB and TolC delineate the IM and OM boundaries, respectively, with the long axis of the pump assembly extending ~320 Å into the periplasm (Fig. 1; Extended Data Fig. 4). This is comparable to the dimensions observed for other types of tripartite assemblies, such as AcrAB-TolC and MacAB-TolC^5-7.

The EmrA hexamer forms a nanochannel with an AssA subdomain for pump assembly

The cryo-EM map revealed five sequentially ordered structural units in EmrA: the singular N-terminal TM helix and the α-helical hairpin, lipoyl, and β-barrel domains, as well as a C-terminal α-helix (Fig. 2a-c; Extended Data Fig. 5). The N-terminal TM helix anchors the four periplasmic units to the IM (Fig. 2b, c). Adaptor proteins, such as AcrA and MacA, utilize a MP domain for interaction with the periplasmic extension of IM transporters^5-7. EmrA and its homologues, as well as the adaptor proteins of T1SS, however, lack such a MP domain (Extended Data Fig. 6; Extended Data Fig. 7). MacA presents a gating ring formed by conserved glutamine residues in the lipoyl domain⁷, which is absent in EmrA. The α-helical hairpin domain structure resembles that of A. aeolicus EmrA, although it is one-third shorter (Extended Data Fig. 6a, b) ¹⁴. The structures of both the lipoyl and β-barrel domains are similar to those of other adaptors (Extended Data Fig. 6; Extended Data Fig. 7). A notable difference is the presence of a unique loop (termed β-CL), comprising residues 294-317, in the β-barrel domain of EmrA (Fig. 2b; Extended Data Fig. 8a), which is conserved among EmrA homologues (Extended Data Fig. 9a). A long coil follows the C-terminus of the β-barrel domain, folding back to engage with the side of the adjacent lipoyl domain in the pump assembly. This coil precedes an α-helix at the terminus (residues 375-390) that, together with the lower portion of the α-helical hairpin, forms a three-stranded coiled-coil, mimicking the α-helical hairpin domain of CusB ²⁷.

The α-helical hairpin, lipoyl and β-barrel domains of EmrA assemble into a hexameric structure reminiscent of AcrA and MacA within the MacAB-TolC and AcrAB-TolC pumps (Fig. 2c; Extended Data Fig. 7) ^5,7. The helical hairpin domains create a nanochannel, referred to as NanoHP. The lipoyl domains form a ring encompassing an internal chamber that extends the nanochannel (named CavLP). The six β-CLs within the β-barrel domains (β-CL1 to β-CL6) vary in conformation, creating a structural unit which we refer to as assembly subdomain A (AssA), which forms the wall of a chamber (named CavEA) (Extended Data Fig. 8a-c). Residues L302 and L303 in the β-CL hexamer form a hydrophobic ring-1 on the IM distal side of CavEA, providing a narrow entryway to CavLP. Conversely, residues I313, V315 and V316 generate a hydrophobic ring, ring 2, on the IM proximal side of CavEA, potentially situated on the surface of the cellular membrane (Extended Data Fig. 8b, c; Extended Data Fig. 9a). The β-CL1 contains an alternative route (termed β-CL1PH) linking the chambers CavEA and CavLP (Extended Data Fig. 8c).

A monomer of EmrB containing an AssB subdomain mediates pump assembly

EmrB, which belongs to the DHA2 family, comprises 14 transmembrane helices, of which H1-H12 adopt the characteristic fold of MFS transporters such as MdfA and EmrD ^16,17. The six-helix bundles, formed by H1-H6 (termed EBN) and H7-H12 (termed EBC), create a V-shaped transporter (Fig. 3a-c) with a pseudo-twofold symmetry axis perpendicular to the membrane plane. Two more TM helices, H_A and H_B, are inserted into the cytoplasmic loop bridging the EBN and EBC domains, but against the periphery of the protein core, similar to YbgH and PepTso ^28-30. The H_A and H_Bcounterparts of QacA and Tet(L) are known to be important for their functions ^19,31.

Structural analysis of EmrB revealed a central aqueous cavity, CavEB_out, located in the core of the membrane that opens toward the periplasmic space, indicating that EmrB adopts an outward-open state (Extended Data Fig. 10a). Entry into the cytoplasm from this cavity is restricted by interactions of side chains between helices H4 and H5 in the EBN domain and helices H10 and H11 in the EBC domain (Extended Data Fig. 10a). Within the central cavity, we located a proton-titratable residue D29, located in motif D, which we hypothesized would be critical in the antiporter function of EmrB ³². This suspicion arose from prior knowledge that the corresponding residues of D34 in the TM1 of QacA and MfdA has previously been shown to be crucial for substrate recognition ^17,18. In our structure, D29 forms a salt bridge with R109 and hydrogen bonds with Q112 and N174, respectively (Fig. 3d). Interestingly, all three of these residues are highly conserved among EmrB homologues (Extended Data Fig. 9b). To verify the importance of these residues in pump function, we performed mutagenesis studies that were predicted to void the function of EmrB. It was found that both EmrB_D29N or EmrB_R109A mutations abrogated bacterial resistance to nalidixic acid, nitroxoline, polymyxin B and neomycin, in support of the involvement of these residues being involved in proton translocation and coupling during transport.

Parts of the 13_th and 14_th TM helices (H11 and H12) of EmrB protrude into the periplasm and form a structural unit named assembly subdomain B (AssB) (Fig. 3a-c). The AssB subdomain is composed of an N-terminal helix X (H_X) following H11, a C-terminal helix Z (H_Z) preceding H12, and a horizontal helix Y (H_Y) parallel to the membrane, along with the loops connecting these helices. The most pronounced difference between EmrB and other MFS transporters is the unique AssB subdomain structure, which is key for pump assembly (Fig. 3b).

Interfacial contacts between EmrA and EmrB

The β-barrel domain of EmrA mediates interaction with EmrB, with the AssA subdomain of EmrA plays a key role through interactions with the periplasmic AssB subdomain of EmrB (Fig. 4a). H_Y in the AssB subdomain penetrates the hydrophobic ring-1 in the AssA subdomain, and H_X and H_Z are positioned along the side of the chamber CavEA. Since H_Y is larger than the narrow entrance formed by hydrophobic ring-1, it becomes trapped above the hydrophobic ring-1, effectively blocking the narrow entrance (Fig. 4b, c; Extended Data Fig. 8b, c). The EmrA_L302Q/L303Q mutation abrogated pump-mediated resistance to nalidixic acid, nitroxoline, polymyxin B and neomycin, indicating that the hydrophobic ring-1 plays an essential role in pump assembly (Fig. 4d; Extended Data Fig. 8c). Below the hydrophobic ring-1, helices H_X and H_Z in the AssB subdomain tilt towards one side of CavEA, making contacts only with β-CL1 to β-CL3 (Fig. 4b). The cavity CavEB_out in EmrB opens to CavEA in the AssA subdomain (Fig. 4c). Given the blockage of the narrow entrance by H_Y, the route of β-CL1PH in β-CL1 serves as the sole link between CavEA and CavLP.

In addition, the cytoplasmic surface of the EmrA β-barrel domains directly engages with the periplasmic region of EmrB, and the N-terminal TM helices of EmrA insert into the membrane, stabilizing the interaction interface. The protruding loops in EmrB, positioned between helices H1-H2, H7-H8 and H9-H10, fit snugly into the concave pockets in the AssA subdomain. The hydrophobic ring-2 in AssA assists in the formation of a sealed continuous channel between CavEB_out and CavEA by resting on the surface of the cellular membrane (Extended Data Fig. 8b, c). The EmrA_{I313Q/V315T/V316T} mutation in hydrophobic ring-2 abrogated bacterial resistance to nalidixic acid, nitroxoline, polymyxin B and neomycin, indicating that the hydrophobic ring-2 is critical for the pump assembly (Fig. 4d; Extended Data Fig. 8c).

Prediction model of EmrB in an inward-open conformation

EmrB has been suggested to adopt an inward-open state for substrate recognition and binding during transport, thereby forming a periplasmic gate ³³. However, in both our cryo-EM structure and all 5 models predicted with high-confidence scores by AlphaFold3, EmrB is consistently displayed in an outward-open state (Fig. 5a; Extended Data Fig. 11a, b) ³⁴. When we utilised the multimer feature of AlphaFold3 to generate a prediction of EmrB in complex with six molecules of EmrA, all 5 of the models generated reveal EmrB in a conformation different from the outward-open state (Fig. 5b; Extended Data Fig. 11c, d). Based on the disappearance of CavEB_outand the presence of a new and enlarged cytosolic cavity (named CavEB_in), we hypothesized that AlphaFold had been able to capture the inward-open state of EmrB previously unobservable in the experimental data set (Fig. 5c) ³³. Using a homology model of EmrB in the inward-open state in conjunction with these predictions, we identified several residues potentially important in the process of translocation. Residues N62, V147, I148 and I288 are all situated along the wall of the cytosolic-facing cavity (Extended Data Fig. 10b). In support of AlphaFold, mutations of N62, V147, and I288 indeed reduced pump-mediated resistance to polymyxin B, neomycin, nitroxoline and nalidixic acid. I148 was crucial for resistance to polymyxin B and neomycin, whereas it appeared dispensable for nitroxoline and nalidixic acid (Fig. 3f). Altogether, these results support AlphaFold's ability to generate testable hypotheses, in addition to demonstrating the importance of specific residues within EmrB in exhibiting poly-specific drug recognition during the transport process.

AF-Cluster reveals the structural transitions of EmrB

Upon close inspection of the structures of EmrB in different states, we detected significant changes in the chemical environments of residues already revealed to be important in our mutagenesis studies. For example, the proposed proton-carrier D29 was shown to establish 3 hydrogen bonds in the outward-open state in comparison to just 2 hydrogen bonds in the inward-open state (Fig. 5d). To provide further evidence that AlphaFold3 had indeed captured the inward-open state and to try gain insight into conformational changes within EmrB, we employed a modified version of AlphaFold2, known as AF-Cluster, to obtain predictions of the various states which EmrB could adopt ³⁵. By grouping the multiple-sequence alignments based on sequence similarity within the MFS family and subsequently performing AlphaFold2 predictions, we captured a plethora of structures representative of the transition between the outward-open to inward-open state of EmrB (Fig. 5e). Interestingly, 75% of these high-confidence predictions could be classified into structures representative of the inward-open states, providing further evidence of this conformation being adopted by EmrB. Furthermore, these predictions also provided insight into the conformational dynamics that EmrB is likely to undergo in shuttling substrates, analogous to the transitions proposed for other members of the MFS family ³³. The helices within the EBC domain appear to remain static, whereas helices H1 – H6 in the EBN domain are suspected to be highly dynamic. The helices appear to pivot at a central point, in support of a ‘rocker-switch’ mechanism of transport for EmrB, common to other MFS antiporters ³⁶. Altogether, these results provide evidence of an inward-open conformation of EmrB, as well as insights into the structural transitions of EmrB that are important in the energy-dependent flux of antibiotics by EmrAB-TolC.

Interactions between EmrA and TolC

A short helix-turn-helix motif located within the α-helical hairpin domain of EmrA mediates tip‐to‐tip interactions with the periplasmic end of TolC, similar to the interactions observed between the homologous MacA and TolC (Extended Data Fig. 12) ⁷. Engagement between TolC and EmrA directly opens the periplasmic aperture of TolC, creating a continuous nanochannel with a width of 25-30 Å, termed NanoTC. TolC is predicted to remain open during the pumping process, as observed previously for RND-type AcrAB-TolC and MexAB-OprM pumps ^5,7,37.

One-step drug transport mechanism of the EmrAB-TolC pump

The structure of the EmrAB-TolC tripartite pump represents the resting apo state structure. The full pump structure reveals that it employs a one-step drug transport mechanism across the entire cell envelope (Fig. 6). EmrA and EmrB possess unique AssA and AssB subdomains, respectively (Fig. 3b; Extended Data Fig. 8b, c). The six β-CLs in AssA vary in conformation, one of which develops a path (β-CL1PH) linking the chambers CavLP and CavEA in the lipoyl and β-barrel domains (Fig. 4b, c; Extended Data Fig. 8a-c). Residues L302 and L303 in the β-CL hexamer form a hydrophobic ring-1, onto which the AssB anchors (Fig. 4; Extended Data Fig. 8b, c). EmrA lacks the MP domain, which enables EmrA and EmrB to form a secure conduit and its hydrophobic ring-2 strengthens this conduit (Fig. 4d; Extended Data Fig. 8b, c). The structure of the EmrAB-TolC pump delineates the complete transport pathway from the central cavity CavEB_out of EmrB to the cell exterior (Extended Data Fig. 13). As a homologue of QacA and MdfA ^17,18,38, EmrB in the IM could adopt an inward-open state to facilitate substrate recognition and binding from the inner leaflet of the IM and cytoplasm, as supported by AlphaFold predictions, homology modelling and mutagenesis studies (Fig. 3f; Fig. 6; Extended Data Fig. 10b). Subsequently, the substrate is transported to the nanochannel formed by EmrA and TolC via an alternating access mechanism, characteristic of MFS transporters (Fig. 6) ³³. The transport pathway involves several essential elements, namely, CavEB_in, CavEB_out, CavEA, β-CL1PH, CavLP, NanoHP, and NanoTC (Extended Data Fig.10a, b; Extended Data Fig. 13). These components form a secure conduit for substrate release to the cell exterior (Fig. 6). The gating helices in EmrB allow substrates to flow against concentration gradients and prevent backflow during operation (Extended Data Fig. 10a, b), resulting in the export of drugs across the IM and OM to the extracellular milieu.

Antibiotic resistance of pathogenic bacteria is a growing clinical problem. Drug efflux pumps in the bacterial cell envelope play important roles in multidrug resistance. In the current study, the E. coli EmrAB-TolC pump was shown to confer multidrug resistance to clinically important antibiotics, including polymyxin B, neomycin and nitroxoline (Extended Data Fig. 1a). The EmrAB-TolC structure identifies key residues that contribute to such multidrug resistance (Fig. 3e, f; Fig. 4d). The conserved hydrophobic ring-1 of EmrA is crucial for the assembly and functioning of the pump and is accessible to the external environment via the open nanochannel in the full pump assembly (Extended Data Fig. 9a; Extended Data Fig. 13); thus, it represents a promising target for the development of drugs that inhibit this type of pumps.

At present, six families of efflux pumps are known to be involved in drug transport: primary transporters of the ATP-binding cassette (ABC) family, and secondary transporters including the major facilitator (MFS), resistance/nodulation/cell division (RND), small multidrug resistance (SMR), multidrug and toxic compounds extrusion (MATE), and proteobacterial antimicrobial compound efflux (PACE) families. In Gram-negative bacteria, all these drug transporters are located in the IM. The members of five families of drug transporters (MFS, SMR, MATE, PACE and ABC) usually function as independent units to translocate substrates across the IM bilayer ⁸. Drugs on the periplasmic side must be further translocated across the OM. Structures of RND-type AcrAB-TolC and ABC-type MacAB-TolC reveal that the IM components AcrB and MacB pick up substrates from the outer leaflet of the IM and periplasm, and efflux them to the extracellular milieu through the tripartite assembly (Extended Data Fig. 14a, b) ^5-7. It is likely that the five transporter systems cooperate with the double membrane-spanning RND-type or ABC-type tripartite pumps to deliver substrates across the OM to the cell exterior. Thus, these tripartite efflux pumps employ a two-step mechanism of periplasmic substrate capture and transport (Extended Data Fig. 14a, b) ^2,8. On the other hand, MFS-type tripartite pumps appear to utilize a one-step mechanism to transport drugs across the entire cell envelope, bypassing the periplasm (Extended Data Fig. 14c). The closely related T1SSs, such as HlyDB-TolC, are believed to employ similar processes for the secretion of protein substrates¹. The HlyDB-TolC pump could form a tripartite assembly. Similar to EmrA, the adapter protein HlyD lacks a MP domain (Extended Data Fig. 6)^4,39, which may enable HlyD and HlyB to form a secure conduit. The ABC family transporter HlyB could drive HlyA across double membranes of the cells via an alternating access mechanism.

The EmrAB-TolC pump differs significantly from AcrAB-TolC and MacAB-TolC pumps. While all three pumps utilize the TolC OM channel, their IM components are derived from distinct transporter families. The periplasmic adaptor proteins have evolved uniquely to complement the specific IM transporters. As a result, each type of tripartite pump utilizes a unique pump assembly and gating mechanism that contributes to its function. The ability of these proteins to recognize and transport various substrates largely depends on the specific domains within their IM transporter components. Understanding the similarities and differences between tripartite multidrug efflux pumps is critical for developing strategies to combat multidrug resistance, a global health challenge.

Data availability

Coordinates have been deposited in the Protein Data Bank (PDB) under PDB codes 8ZAL (EmrAB-TolC pump-EA) and 8ZAR (EmrAB-TolC pump-FA). Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMD) under EMD codes EMD-39879 (EmrAB-TolC pump-EA) and EMD-39885 (EmrAB-TolC pump-FA).

Acknowledgements:

This work was supported by the National Key R&D Program of China (2022YFC2303200); National Natural Science Foundation of China (31971133 to D.D.; 32270064 to Y.C.), the Science and Technology Commission of Shanghai Municipality (19PJ1407900, 19JC1414000 and 22WZ2504100 to D.D.; 21ZR1471300 and 2019SHZDZX02 to Y.C.), and the Chinese Academy of Sciences (176002GJHZ2022022MI to Y.C.). BFL was supported by ERC Advanced grant (742210) and a Wellcome Trust Investigator award (200873/Z/16/Z), and MLJ by a UKRI Medical Research Council (MRC) Intramural Programme Award MC_UU_00025/4 (RG94521). Cryo-EM data were collected at the Bio-Electron Microscopy Facility of ShanghaiTech University with the assistance of Q. Sun, D. Liu, Z. Zhang, L. Wang and Y. Yang. We thank the Molecular Imaging Core Facility, the Molecular and Cell Biology Core Facility, and the Multi-Omics Core Facility at the School of Life Science and Technology for providing technical support. We are also grateful for the support of Lajos Kalmar of the MRC Toxicology Unit in the use of high-performance computing used in this study.

Author contributions:

Z.Z. performed cloning and overexpression of the EmrAB-TolC complex; Z.Z. and T.M. purified the EmrAB-TolC complex, prepared cryo-EM samples, collected cryo-EM data, determined structures, performed model building and refinement and prepared figures for the manuscript; X.G., W.S., H.Z., J.G., S.L. H.L. and Q.O. optimised in-column peptide-disc methods, prepared homemade graphene monolayer grids, and assisted the collection of cryo-EM data; R.D., H.J., Z.Z. and T.M. performed antibiotics sensitivity assays; Y.C. supervised antibiotics sensitivity assays and discussed project design; M.L.J. performed protein structure predictions and modelling;D.D. and B.L. conceived the project; D.D. designed and supervised all experiments; D.D. and B.L. wrote the manuscript. All the authors contributed to the data interpretation and manuscript preparation.

Declaration of interests:

The authors declare no competing interests.

Construction of EmrAB-TolC complex overexpression vectors

The BRIL gene was synthesized by AZENTA (Suzhou, China) and amplified using the brilpet-F and bril-link-R primers. The emrA, emrB, tolC, and acrA genes were amplified from genomic DNA of E. coli K-12 strain MG1655. The emrB (37-1494 bp) genefragmentwas amplified using primers emrB-link-F and emrBGS-R. The full-length emrA gene was first amplified using primers emrAGS-F and emrApet-R; the pETDuet-1 plasmid was linearized using primer pair Petduet_F and Petduet_R; and the BRIL, emrB, and emrA DNA fragments were inserted into the pETDuet-1 vector using NovoRec plus One step PCR Cloning Kit (Novorprotein, Shanghai, China), resulting in pETDuet-BRIL-emrB-polyGlySer-emrA-6His. The ALFA-tag sequence was subsequently inserted into the 3′ end of emrB by site-directed mutagenesis, generating pETDuet-BRIL-emrB-ALFA-polyGlySer-emrA-6His (abbreviated as pET_BA).

Using primers TolCinf_F and TolCFLAGXhoI_R, the tolC gene was amplified, and the product served as a template for a second amplification using primers TolCinf_F and TolCFLAG_inf_R. The resulting tolC-FLAG DNA fragment was inserted into pRSFDuet-1 (digested with NdeI) using the in-fusion ligation method of the NovoRec plus One step PCR Cloning Kit (Novorprotein, Shanghai, China), thereby generating pRSFDuet-tolC-FLAG. The emrA DNA fragment was amplified using primers emrAinsert_F and emrAinsert_R and inserted into the pRSFDuet-tolC-FLAG vector using the in-fusion cloning method (Novorprotein, Shanghai, China), resulting in pRSFDuet-emrA-tolC-FLAG (abbreviated as pRSF_AC).

Primer pair ΔemrAinsert_F and emrAinsert_R was used to amplify the truncated ΔemrA gene (142-1173 bp), and the ΔemrA DNA fragment was inserted into the pRSFDuet-tolC-FLAG vector using the In-Fusion cloning method (Novorprotien, Shanghai, China), generating pRSFDuet-ΔemrA-tolC-FLAG. The N-terminal 81 bp region of acrA (acrAsignal) was amplified using the acrAs_F and acrAs_R primer pair, and the resulting acrAsignal DNA fragment was inserted into the 5' end of ΔemrA in pRSFDuet-ΔemrA-tolC-FLAG using the In-fusion cloning method (Novorprotien, Shanghai, China), generating pRSFDuet-acrAsignal-ΔemrA-tolC-FLAG (abbreviated as pRSF_ΔAC).

The emrAB genes were amplified from genomic DNA of E. coli K12 strain MG1655 using primers EmrAB_F and EmrAB_R, the pETDuet-1 plasmid was amplified using primers Petduet_V_F and Petduet_V_R, and the emrAB fragment was inserted into pETDuet-1 using an In-Fusion Ligation Kit, resulting in pETDuet-emrAB.

The site-directed mutants EmrAB_D29N and EmrAB_R109A were produced using pETDuet-emrAB as a template with the primer pairs emrB_D29N_F/emrB_D29N_R and emrB_R109A_F/emrB_R109A_R, resulting in pETDuet-emrAB_D29Nand pETDuet-emrAB_R109A.

The site-directed mutants EmrAB_N62A, EmrAB_V147A, EmrAB_V148A, and EmrAB_I288A were created by using pETDuet-emrAB as a template with the primer pairs emrB_N62A_F/emrB_N62A_R, emrB_V147A_F/emrB_V147A_R, emrB_V148A_F/emrB_V148A_R, and emrB_I288A_F/emrB_I288A_R, resulting in pETDuet-emrAB_N62A, pETDuet-emrAB_V147A, pETDuet-emrAB_V148A, and pETDuet-emrAB_I288A.

The site-directed mutants EmrA_L302Q/L303QB and EmrA_{I313Q/V315T/V316T}B were produced using pETDuet-emrAB as a template with the primer pairs emrA_L302Q/L303Q_F/emrA_L302Q/L303Q_R and emrA_{I313Q/V315T/V316T}_F/ emrA_{I313Q/V315T/V316T}_R, resulting in plasmids pETDuet-emrA_L302Q/L303QB and pETDuet-emrA_{I313Q/V315T/V316T}B.

The enzyme used to introduce the mutations above-mentioned was purchased from CloneAmp HiFi PCR Premix (Clontech, Germany). Then the modified plasmids above-mentioned were amplified and extracted using SPARKeasy Mini Plasmid Ultra-Fast Kit (Shandong Sparkjade Biotechnology Co.,Ltd.).

Primers used to generate all constructs are listed in Extended Data Table 1.

Overexpression and purification of E. coli EmrAB-TolC

The plasmids pET_BA and pRSF_ΔAC were used to co-express and purify EmrAB-TolC pump-EA, which contains the EmrB-EmrA fusion protein, TolC and a modified free EmrA with the N-terminal residues 1-47 replaced by residues 1-27 of AcrA. The E. coli C43 (DE3) ΔacrAB strain was transformed with plasmids pET_BA and pRSF_ΔAC. From an agar plate containing appropriate antibiotics, a single colony was picked and inoculated into 20 mL of LB medium supplemented with 50 μg mL^-1 kanamycin and 100 μg mL^-1 carbenicillin in a 50 mL centrifuge tube. The culture was incubated at 37°C with shaking at 220 rpm for 4-5 h.

A 10 mL culture aliquot was then transferred to 1 L of 2´YT medium containing antibiotics in a 2 L baffled flask. The same incubation conditions were applied. Cells were cultured until a density (absorbance at 600 nm, A₆₀₀) of 0.5-0.6, induced with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG), the temperature was reduced to 20°C, and culturing was continued overnight.

The cells were harvested by centrifugation, and the cell pellet from 6 L of culture was resuspended in 200 mL of lysis buffer containing 20 mM Tris (pH 8.0) and 300 mM NaCl. Afterwards, 2 mL of 100´EDTA-Free Protease Inhibitor Cocktail (APExBIO, USA) was added to a final concentration of 1´. Lysozyme and DNase I were added to final concentrations of 5 mg/mL and 5 U/mL, respectively. The cell mixture was incubated at 4°C for 1 h before high-pressure homogenization via three passages at 15,000 psi at 4°C. The lysate was centrifuged to remove cell debris, and the supernatant was ultracentrifuged to pellet the cellular membranes.

Lysis buffer was used to resuspend the membrane pellet. EDTA-free protease inhibitor cocktail (100´) was added to a final concentration of 1´, n-dodecyl-β-D-maltoside (DDM) was added to a final concentration of 1.5%, and the mixture was gently stirred at 4°C for 3 h. The mixture was ultracentrifuged. Imidazole was added to the supernatant to a final concentration of 20 mM, and the mixture was applied to a 1 mL HiTrap Chelating column (Cytiva, USA) charged with Ni^2+.

The column was washed with lysis buffer containing 0.05% DDM and 50 mM imidazole, followed by lysis buffer supplemented with 0.01% DDM. Peptidisc ^40,41 (1 mL) at a concentration of 5 mg/mL in lysis buffer was injected onto the column and incubated at 4°C for 1 h. The column was washed with 10 mL of lysis buffer containing 1 mg/mL peptide disc. Elution of the 6´His-tagged EmrAB-TolC protein complex was achieved with lysis buffer containing 300 mM imidazole. Buffer exchange of the eluate into Buffer-I, which consisted of 20 mM Tris (pH 7.5) and 150 mM NaCl, was conducted using a HiTrap Desalting column (Cytiva, USA). The 6´His-tagged EmrAB-TolC complex was further purified using ANTI-FLAG M2 affinity resin (GenScript, Nanjing, China). The resin was prepared by sequential washing with glycine HCl (pH 3.5) and Buffer-I. The protein solution and resin were mixed, gently rotated for 1 h at 4°C, loaded into a chromatography column, and washed with Buffer-I. The mixture was resuspended in 1 mg/mL FLAG-peptide in Buffer-I, incubated for 30 min, centrifuged, and the supernatant was loaded onto a mini chromatography column to remove residual resin. This step was repeated three times. Fractions containing EmrAB-TolC were pooled, concentrated using a centrifugal filter unit (Merck, Germany) with a molecular weight cut-off of 100 kDa, flash-frozen in liquid nitrogen and stored at -80°C.

The plasmids pET_BA and pRSF_AC were used to co-express and purify the EmrAB-TolC pump-FA containing the EmrB-EmrA fusion protein, TolC, and wild-type free EmrA using the same procedure employed for the pump-EA.

Cell growth for the antibiotic sensitivity assay

Single colonies of E. coli C43(DE3) ΔacrAB harboring the plasmids pETDuet/pRSFDuet, pET_BA/pRSF_AC, pET_BA/pRSF_ΔAC, pETDuet-emrAB/pRSFDuet-tolC-FLAG, pETDuet-emrAB_D29N/pRSFDuet-tolC-FLAG, pETDuet-emrAB_R109A/pRSFDuet-tolC-FLAG, pETDuet-emrA_L30Q/L303QB/pRSFDuet-tolC-FLAG, pETDuet-emrA_{I313A/V315A/V316A}B/pRSFDuet-tolC-FLAG, pETDuet-emrAB_N62A/pRSFDuet-tolC-FLAG, pETDuet-emrAB_V147A/pRSFDuet-tolC-FLAG, pETDuet-emrAB_V148A/pRSFDuet-tolC-FLAG, or pETDuet-emrAB_I288A/pRSFDuet-tolC-FLAG were grown in fresh LB supplemented with 50 µg/mL kanamycin and 100 µg/mL ampicillin at 37℃ with shaking at 220 rpm. When the optical density at 600 nm (OD₆₀₀) reached 0.5–0.6, expression was induced with 100 µM IPTG, the temperature was reduced to 22°C, and the incubation was continued for 1 h.

The cells harboring pET_BA/pRSF_AC or pET_BA/pRSF_ΔAC were harvested by centrifugation, after which the cell pellets were resuspended in fresh medium to an OD₆₀₀ of 0.1 and treated with 0.8 mg/mL nitroxoline and 30 µM IPTG. Cell growth was monitored over time by measuring the OD₆₀₀ at 37°C in a Spark microplate reader (Tecan, Austria).

The cells harboring the remaining constructs were harvested by centrifugation, resuspended in fresh medium to an OD₆₀₀ of 0.5, and 10-fold serially diluted. Then 5 µL of each culture was plated on LB agar containing 60 µM IPTG in the presence of 1 µg/mL nalidixic acid, 5 µg/mL nitroxoline, 1 µg/mL polymyxin B, or 1 µg/mL neomycin. As a control, 5 μL of all suspensions were plated on LB agar without antibiotics. Cell growth was monitored after culture at 37°C overnight. The assay was performed three times independently.

Electron microscopy data collection

For the EmrAB-TolC pump-EA cryo-EM assays, 3.5 μL aliquots of purified protein in peptidisc (protein concentration = 0.7 mg/mL) were added to glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh; Quantifoil Micro Tools GmbH). Blotting was performed with filter paper for 3.5 s to remove excess sample, and a Vitrobot Mark IV instrument (Thermo Fisher Scientific) was used for rapid freezing in a liquid ethane slush. A Titan Krios electron microscope (Thermo Fisher Scientific) operating at 300 kV coupled with a SerialEM and a Gatan K3-Summit detector (Gatan, Inc.) operating in super-resolution counting mode ⁴² were used to automatically collect zero-energy-loss images of frozen and hydrated grids. Using a slit width of 20 eV, a GIF-Quantum energy filter (Gatan) was applied to exclude inelastically scattered electrons. Using a dose rate of ∼15.15 electrons Å^-2 s^-1 (~18 electrons pixel^-1 s^-1) at an adjusted magnification of ´45,871.6 (yielding a pixel size of 1.09 Å at the sample level) and a total dose of ∼60 electrons Å⁻² at the sample, 60 movie frames were recorded. The final dataset comprised 4334 movie stacks with defocus values between -1.0 and -2.5 μm.

For the EmrAB-TolC pump-FA, a holey carbon grid (Quantifoil Au R1.2/1.3, 300 mesh) was overlayed with a homemade graphene monolayer and cleaned with UV/ozone at room temperature for 10 min using a Gatan SOLARUS (950) Plasma Cleaning System (Gatan, Inc.), ensuring hydrophilicity of the graphene grid. Next, 3.5 μL aliquots of the purified protein sample from the peptidisc (0.1 mg/mL concentration) were applied to the grid and incubated for 30 s. Excess sample was removed by blotting with filter paper for 3.5 s, followed by rapid freezing in liquid ethane slush using a Vitrobot Mark IV instrument. Frozen-hydrated EmrAB-TolC particles were subjected to automatic data collection using a Titan Krios electron microscope at 300 kV with SerialEM and a Gatan K3-Summit direct electron detector running in super-resolution counting mode at an adjusted magnification of 47,169.9, equating to a measured physical pixel size of 1.06 Å and a dose rate of ∼16 electrons Å^-2 s^-1(∼18 electrons pixel^-1 s^-1). Exposures lasting 3.533 s were split into 60 movie frames, resulting in an accumulated dose of ∼56.6 electrons Å^-2 at the sample. Using a defocus range of -1.0 to -2.5 μm, 3306 movie stacks were collected.

Image processing

For the EmrAB-TolC pump-EA, the super-resolution movie frames were adjusted, including correction for gain reference and 2´ binning, followed by motion correction using MOTIONCORR2 ⁴³. Merging of the aligned movie frames into micrographs then allowed estimation of the contrast transfer function (CTF) using CTFFIND4 ⁴⁴. RELION v3.1.3 software was used for subsequent image processing steps^45,46. Templates for automatic particle picking were derived from (reference-free) two-dimensional classification of a manually selected particle subset. To minimize reference bias, these templates underwent low-pass filtering to 20 Å, enabling the automatic selection of 816,894 particles from all the micrographs. Two-dimensional classification of the images yielded 480,902 suitable particles.

Additional image processing steps were carried out using CryoSPARC ⁴⁷. Particles chosen via two-dimensional classification within RELION were imported into CryoSPARC and subjected to two further rounds of two-dimensional classification to discard any obviously discrepant particles, yielding 56,006 particles for classification by ab initio reconstruction using CryoSPARC. The parameters for this classification included two-class ab initio reconstruction with specific settings (initial alignment resolution of 25 Å; maximum alignment resolution of 6 Å; initial minibatch size of 150; final size of 600; and class similarity of 0) ⁴⁸. A total of 33,460 particles contributed to the resulting 3D volume, which was subjected to nonuniform refinement, generating an EmrAB-TolC reconstruction at a resolution of 3.14 Å. The resulting map quality for the EmrA and TolC sections was high, but the quality for the EmrB section was low. The AlphaFold ⁴⁹-generated EmrB model was subsequently transformed into a density map using e2pdb2mrc.py in EMAN2 ⁵⁰and subsequently aligned to the EmrB density map section within the EmrAB-TolC reconstruction. A focused mask on EmrB was applied to carry out 3D classification (without alignment), yielding five quality classes from 10. Aligning the 3D volumes and corresponding 19,180 particles from these classes using the Align 3D Maps program in CryoSPARC resulted in a homogeneous reconstruction of EmrAB-TolC at a resolution of 3.45 angstroms, with an improved map quality for the EmrB section. Local refinement was then carried out using a focused mask on the EmrAB portion, leading to a refined EmrAB-TolC reconstruction at 3.11 Å with improved map quality for the EmrAB sections (map-1 of pump-EA; and Extended Data Fig. 2c, d). Local refinement was also conducted using a focused mask on the TolC and α-helical hairpin domains of EmrA, leading to a refined reconstruction at 3.14 Å with better map quality for the EmrA-TolC sections (map-2 of pump-EA; and Extended Data Fig. 2c, d).

A similar image processing procedure was employed for EmrAB-TolC pump-FA, which generated a reconstruction at 3.59 Å resolution (Extended Data Fig. 3c, d; Extended Data Table 2).

Resolution estimation was accomplished via CryoSPARC using independently refined half-reconstructions. The criterion for Fourier shell correlation was set at 0.143. The ResMap wrapper in CryoSPARC was used to calculate variation in local resolution (Extended Data Fig. 2; Extended Data Fig. 3) ⁵¹.

Protein structure prediction and modelling

AlphaFold3, AlphaFold2 and AF-Cluster were all employed for protein structure predictions. High-confidence predictions were selected based on their per-residue confidence score and corresponding predicted aligned error plot. Unless stated otherwise, the top rank of 5 predictions is shown and is visualized in ChimeraX Version 1.7. AlphaFold3 Beta was used to generate models of full-length EmrB (UniProt P0AEJ0) ³⁴. AlphaFold3-Multimer Beta was used to generate models of EmrAB, consisting of one molecule of full-length EmrB (UniProt P0AEJ0) with six molecules of full-length EmrA (UniProt P27303). AF-Cluster was used to predict multiple conformations of EmrB ³⁵. An MSA of EmrB (UniProt P0AEJ0) consisting of ~6000 sequences was first generated using ColabFold ⁵². These sequences were then clustered into ~300 groups based on similarity using DBSCAN. A total of 16 clusters of sufficient size (at least 30 sequences) were then subsequently predicted using AlphaFold2. These conformations were visualized and analyzed using both ChimeraX and ChimeraUCSF.

Model building and refinement

The two half-maps of cryo-EM map-1 for EmrAB-TolC pump-EA were used to perform local density sharpening with LocSpiral ⁵¹. The resulting full map was segmented into three TolC protomers, six EmrA protomers, and one EmrB protomer. A model of the β-barrel domain without β-CL, and the lipoyl domain, generated by AlphaFold, was fitted to the density map of individual EmrA protomers using Chimera. The α-helical hairpin domain, C-terminal α-helix, and β-CL of the β-barrel domain were manually built using Coot ⁵³. Each EmrA protomer model was subsequently refined with Rosetta ⁵⁴.

A homology model of EmrB in the inward-open state was generated by AlphaFold3 ³⁴. Chimera⁵⁵ was used to fit the EBC domain of this homology model to the density map. Models of the AssB subdomain and EBN domain of EmrB were manually built using Coot. The complete EmrB model based on the density map was subsequently refined using Rosetta.

The two half-maps of cryo-EM map-2 for EmrAB-TolC pump-EA were used to conduct local density sharpening using LocSpiral. The structure of trimeric TolC, derived from the MacAB-TolC pump (PDB code: 5NIK), was docked into the TolC section of the density-modified map-2 using Chimera. To improve the local fit to the density, manual adjustments were made to the model using Coot.

Models of individual components were fitted into the cryo-EM map-1 of EmrAB-TolC using Chimera. The cryo-EM map-1 of EmrAB-TolC pump-A without global B-factor sharpening was used to perform model-based local density sharpening with LocScale ⁵⁶. The entire EmrAB-TolC pump-EA model underwent real-space refinement against this density-modified map in the Phenix package ⁵⁷.

The refined model of EmrAB-TolC pump-EA was fitted into the cryo-EM map of pump-FA. The model section of the N-terminal TM helix of EmrA, generated by AlphaFold, was fitted into the density map of the individual EmrA protomer. Model sections without defined density maps were deleted. Model-based local density sharpening of the cryo-EM map of EmrAB-TolC pump-FA without global B-factor sharpening was carried out using LocScale (53). The entire EmrAB-TolC pump-FA model was refined using real-space refinement via the Phenix package.

Any Ramachandran outliers were manually corrected in Coot, and stereochemistry was ensured using MolProbity (Extended Data Table 2)⁵⁸.

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One-step drug transport across two membranes of Gram-negative bacteria

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