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Allosteric drug transport mechanism of multidrug transporter AcrB

Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.

G ram-negative bacteria comprise a double-layered envelope, the inner and outer membrane (IM and OM) enclosing a compartment known as the periplasm. These bacteria are intrinsically resistant against cytotoxic substances due to the synergistic action of the IM and OM and a network of multidrug efflux systems 1 . Various efflux pumps with broad overlapping drug preferences sequester and transport drugs across the IM to supply the highly polyspecific tripartite resistance-nodulation-cell division (RND) efflux pump systems with compounds to be extruded across the OM barrier 2,3 . In clinical Gram-negative strains, RND efflux pumps are often upregulated and contribute to the overall multidrug resistance phenotype, leaving infections untreatable with our current arsenal of antibiotics 4 . AcrAB-TolC is the main drug efflux system in Escherichia coli (Fig. 1a) 2,5 . AcrB is the homotrimeric IM protonmotive force-driven H + /drug antiporter component (Supplementary Note 1.1) which recognizes and actively transports multiple drugs (antibiotics, detergents, dyes, and solvents) via the tightly connected AcrA and TolC channels across the OM, leading to an observable resistance phenotype (Fig. 1a) [6][7][8][9][10][11][12][13][14][15][16][17] . Four different entry channels (CH1-CH4) have been described with putative entrances from the periplasm or the outer leaflet of the IM toward the two drug-binding pockets, the access pocket (AP) and deep binding pocket (DBP) inside the AcrB pump (Fig. 1b, c) 8,9,[17][18][19] . For one of these observed channels, CH3, specificity towards planar aromatic cationic drugs has been postulated on basis of mutational analysis of the CH3 entrance region and by blocking the path between the AP and DBP 18 . The drug specificity of the other channels is elusive, but binding of erythromycin, rifampicin 10 , and a doxorubicin-dimer 12 to the AP indicates that CH2 prefers high molecular weight (HMW) drugs, and CH4 was postulated to transport carboxylated drugs 19 (Fig. 1). The transport of drugs through the channels has been postulated to be mediated by a mechanism resembling the action of a peristaltic pump 8 . Moreover, the efflux of certain drugs was shown to be enhanced in the simultaneous presence of other substrates, indicative of a kinetically cooperative transport mechanism 20 .
In this work, we address three main questions: (1) How are drugs transported through the channels (CH1-CH4) towards the drug-binding pockets (AP and DBP) and is there a drug specificity for each channel? (2) Is the transport mechanism different for high molecular weight (HMW) drugs like macrolides and ansamycins compared to low molecular weight (LMW) compounds like β-lactams, detergents and dyes? And (3) what are molecular determinants of the observed cooperativity in the presence of more than one drug? 20 We present co-structures of AcrB in different intermediate transport states on basis of which we conducted extensive mutational analysis combined with drug susceptibility and thiol cross-linking studies. The results suggest that drugs are transported via dedicated transport pathways through AcrB depending on their physicochemical properties, which results in a broad polyspecific drug resistance phenotype by the action of a single pump. We also found that AcrB comprises a deep transmembrane binding pocket for fusidic acid, oxacillin, or novobiocin. We suggest that it might act as an allosteric binding site, facilitating binding of other drugs to other sites and enhancing the catalytic efficiency of drug efflux.

Results and discussion
Substrate access from the membrane towards the deep binding pocket (DBP). The transmembrane domains (TMDs) of the AcrB L and T protomers comprise two grooves (TM1/TM2 and TM7/ TM8) which serve as putative entrance sites to channel CH1 and CH4, leading substrates to the access pocket (AP) or deep binding pocket (DBP) inside the porter domain (PD) 9,10,12,18,19,21 (Fig. 1). In several crystal structures, the AcrB substrate dodecyl β-Dmaltoside (DDM) is apparent at the TM7/TM8 groove entrance site of CH1 of the L protomer 9,12,19,22 (Fig. 2a-c). Here, we present a crystal structure of the asymmetric LTO AcrB trimer (in complex with designed ankyrin repeat proteins (DARPins) 9,12 as crystallization chaperones, used for all structures presented) with the L protomer in a L-to-T transition state (L2 protomer, see  Table 1 for differences between L and L2). A DDM molecule is trapped in a transient L to T state of the CH1 tunnel (which we designated TM8/PC2 tunnel, see Supplementary Note 1.2) towards the AP (Fig. 2a, b). The DDM maltose moiety is situated at the AP, proximal to a small flexible loop which separates the AP and the DBP (switch loop, Fig. 1c) 12 and the closed DBP. DDM is sandwiched between Leu674 and Leu828 and forms hydrogen bonds with Asn719 and Asp681 (Fig. 2b). The DDM acyl chain localizes at the interface of the PC2 subdomain (in the PD, Fig. 1c) and the TM7/TM8 groove (in the TMD, Fig. 1c) and has several van der Waals interactions with non-polar side chains of the tilted TM8 and with a flexible loop which connects the PC1 and PC2 subdomains (the c-loop, Fig. 1c and Fig. 2b, c). A most prominent difference is the large conformational change of the c-loop, moving from a TM8 proximal orientation to a distal one (Fig. 2c). This intermediate conformation is in support of the suggested peristaltic pump hypothesis 8 , rather than by diffusion of drugs through the observed tunnels towards the AP and/or DBP. A comparison with two other structure snapshots 12,22 featuring DDM in the TM7/ TM8 groove a rather clear picture emerges of the peristaltic movement of DDM toward the observed intermediate binding site (Fig. 2c). Residues lining the transient CH1 pathway were systematically substituted by Ala and the substitution-variants analyzed via drug susceptibility tests with 14 different drugs (Supplementary Data 1a, b; Supplementary Fig. 7a, b; Supplementary Note 1.3). Substitution of residues facing the inside of the TM7/TM8 groove and those residing in the entrance area of CH1, i.e. Ile38 23 , Ile466, Leu393, Phe563, Ile671, or Leu674 (the latter also in combination with substitutions in juxtaposed Asp681, Asn719, and Met862) show a most prominent effect on susceptibility for drugs with low molecular weight (LMW) and low polar surface area (LPSA), such as β-lactams, linezolid, and phenicols. Susceptibility tests furthermore implicate that the transient state of CH1 is specific for DDM ( Supplementary  Fig. 1b) as simultaneous Ala-substitution of both DDMinteraction partners, Phe563 and Leu674 (Fig. 2b), results in complete sensitivity towards DDM (with the Asp407Asn substitution variant as negative control 24 ). Sensitivity towards polyaromatic cationic drugs or to a lesser extent also erythromycin, on the other hand, was unchanged to all 40 out of 42 substitution variants compared to cells harbouring wild-type AcrB (Supplementary Data 1b; Supplementary Fig 7b), indicating that polyaromatic cationic drugs and erythromycin take other paths through AcrB (through CH2 and CH3, Fig. 1b).
The Phe563 side chain at the entrance to CH1 appears to act as a guarding residue for further CH1 entry 21 ( Supplementary  Fig. 1a  we realized that the c-loop may play an important role in drug transport not only via CH1 but also via CH4 ( Fig. 2c; Supplementary Fig. 1e; Supplementary Note 1.6). We therefore compromised the entrances to both CH1 and CH4 (Phe380A-la_Phe563Ala), and observed indeed a strong sensitivity towards FUA, β-lactams, and the failure to grow on DDM by cells harbouring this variant (Supplementary Data 1c; Supplementary  Fig. 1b; Supplementary Fig. 7c), whereas wild-type activity is retained towards doxorubicin and rhodamine 6 G (R6G), presumably transported by the unaffected CH3 18 ( Fig. 1b; Supplementary Data 1c; Supplementary Fig. 7c). CH4 transport-compromised AcrB-Phe380Ala shows binding of FUA at the lower end of the TM7/TM8 groove proximal to the cytoplasmic side ( Fig. 2d; Supplementary Fig. 1f), surrounded by TM5 and TM7-TM10. This implies that FUA may enter via CH1, but also via CH4 19 and is in line with the observed substitution phenotypes (Supplementary Data 1c; Supplementary Fig. 7c). In sum, the results suggest that CH1, with its TM7/TM8 entrance groove, is the main transport pathway for LMW/LPSA drugs, DDM, as well as FUA and novobiocin, starting with binding at the TM7/TM8 groove and peristaltic movement of the drugs to the DBP ( In an attempt to prevent ansamycin binding, we made the switch loop more rigid by Gly619 and/or Gly621 to Pro substitutions. Surprisingly, the crystal structure of these variants showed propensity to bind either 3-FOR to both L protomer AP and the smaller AP in the T protomer in the same AcrB trimer (  homolog RND pump from Neisseria gonorrhoeae 26 . During the T to O transition, these drugs are then exported via the DBP area through the O protomer exit tunnel (Fig. 5). The binding of multiple substrates 10,12 ( Fig. 4b and Supplementary Fig. 2a-f) suggest a possible role in the observed cooperativity in presence of more than one drug 20,27 . Hence, we started to experiment with antibiotic cocktails to observe multidrug binding to AcrB.
Transmembrane domain allosteric substrate binding. We used several drug combinations for co-crystallization assays, and cocrystallization of wild-type AcrB with an antibiotic cocktail of erythromycin, linezolid, oxacillin, and FUA (1 mM each) led to the elucidation of a co-structure with FUA bound to a deep concave crevice surrounded by TM2, TM4, TM10-12, and near the Asp407/Asp408/Lys940 proton relay residues [6][7][8][9] (Fig. 6a,  . This was unexpected, since the TMDs of the AcrB protomers are known thus far to exclusively facilitate the transport of protons via the proton relay residues 3,15 . The structure displays a substantial displacement of TM11 and TM12 (~13.6°and~36.7°tilting, respectively, Fig. 6a) towards the periphery of the TMD in the T protomer creating a~1700 Å 3 cavity to accommodate FUA (we call this cavity the deep TMD binding pocket, TMD-BP) (Fig. 6a, b and Supplementary Fig 4b).
No major conformational changes are observed in the L and O protomer (Supplementary Table 1a). Interestingly, two further FUA molecules bind to this T protomer as well. One FUA binds to a site akin to the previously reported TM1/2 groove site 19,25 ( Supplementary Fig. 2j, k). The other FUA molecule binds inside the TM7/TM8 groove (Fig. 2e), similar to the binding of FUA to the Phe380Ala variant (as discussed above, Fig. 2d), but deeper into the TMD core (Fig. 2f). Most peculiar, under FUA-only cocrystallization conditions leading to binding of FUA only to the TM1/TM2 groove 25 , high FUA concentrations (4 or 5 mM) were  mandatory. In the structure presented here, FUA, erythromycin, oxacillin, and linezolid were only present at 1 mM concentration each, and we observe binding of FUA at the TM1/TM2 groove, the TM7/TM8 groove and TMD-BP. These observations suggest allosteric drug binding, and therefore the observed TMD-BP is proposed to be an allosteric site. A structure derived from AcrB co-crystallization with a mixture of β-lactams (dicloxacillin, oxacillin, and piperacillin), resulting in the structure with the DDM-bound L2 protomer (see above) displays a similar, albeit less extensive movement of TM11 in the T protomer. This results in a notable, but smaller cavity of~304 Å 3 within the T protomer ( Supplementary Fig. 4c). We propose that the latter structure represents a state prior to binding of drug in the TMD-BP (partially induced vs. fully induced in case of drug binding). The putative TMD-BP was probed using MTS-rhodamine (MTS-R), a thiol-reactive rhodamine crosslinker 25 . Cys-substitution of Ala981 in an otherwise active Cys-less AcrB background 28 was selected for probing AcrB variants. The Ala981Cys variant showed no compromised activity to all the tested drugs (Supplementary Data 2c; Supplementary Fig. 7f). MTS-R, but not N-(1-pyrenyl) maleimide (P-MAL) 29 reacts efficiently with Cys981 (K d app = 2.68 μM, Supplementary Fig. 4e), indicating that the putative allosteric TMD-BP is specific towards MTS-R. A concentration-dependent reduction of MTS-R labelling of C981 in presence of FUA, oxacillin, or novobiocin (K i app = 38, 446, or 95 μM, respectively) is observed (Fig. 6c). Linezolid and chloramphenicol were unable to protect against MTS-R labelling, whereas erythromycin induced a concentration-dependent increased labeling of Cys981 ( Supplementary Fig. 4f). The latter observation might be due to erythromycin-specific TMD alteration of AcrB and thereby facilitation of MTS-R binding.
Substitution of TMD-BP residues in contact with FUA such as Leu400 (TM4) results in severe increased susceptibilities for phenicols, β-lactams, linezolid, FUA, erythromycin, and to a much lesser degree novobiocin (Supplementary Data 2c; Supplementary Fig. 7f); however, there is no change in susceptibility towards doxorubicin and R6G. Thr934Ala and Leu937Ala (both TM10) show severe susceptibility effects for β-lactams, FUA, novobiocin, and erythromycin, moderate for phenicols (Thr934Ala only), and linezolid (Supplementary Data 2c; Supplementary Fig. 7f). Doxorubicin, R6G, and tetraphenylphosphonium (TPP) susceptibilities were not (Thr934Ala) or mildly to moderately (Leu937Ala) affected. These complex phenotypes can be viewed in the line of (a) a general loss of activity, which can be excluded since doxorubicin and R6G (and for most substitutions also TPP) are showing in most cases wild-type susceptibilities (Supplementary Data 2c; Supplementary Fig. 7f). A second interpretation (b) assumes that the observed TMD-BP is a substrate binding site for the drugs to access the TM1/TM2 or TM7/TM8 grooves, from where the drugs are further transported to the drug-binding AP and DBP. A third option (c) is to assume an allosteric binding site (Fig. 7). We only observed the binding of additional FUA molecules to both of the TM1/TM2 or TM7/TM8 grooves in case FUA was bound to the TMD-BP. Therefore, binding of drugs to the TMD-BP might facilitate initial binding of other drugs to other sites. Furthermore, the TMD-BP exposes a large cavity accessible from the periplasmic side towards the Asp407/Asp408/Lys940 proton relay residues. Binding of drugs near these residues and tilting of TM11/12, as observed in the FUA-bound structure, might be a facilitator for the binding of protons to the Asp407 and/or Asp408 protonation sites or the subsequent conformational change after proton binding, leading to a strong increase in pump activity. This allosteric hyperactivation of the pump might be suitable in case drug concentrations reach levels affecting the integrity of the IM, which would impede the survival of the cell. Since drug efflux pumps like AcrAB-TolC consume a considerable amount of energy (proton-motive force) in presence of drugs 25 , allosteric regulation of its activity might represent a resource-saving measure.
Our results highlight the flexibility of the AcrB RND transporter to recognize, bind, and transport multiple drugs. It employs multiple binding sites and pathways comprising different substrate specificities resulting in the observed polyspecific drug resistance phenotype. Moreover, the H + -conducting transmembrane domain of AcrB might include an allosteric TMD-BP which, if occupied with drug, may cause hyperactivation or even cooperative multidrug transport.

Methods
Bacterial strains and growth conditions. E. coli MachT1 (Life Technologies) cells were routinely used for cloning. E. coli C43 (DE3) 30 ΔacrAB harbouring pET24acrB His was routinely grown in LB medium in the presence of 50 μg ml −1 kanamycin at 37°C 31,32 .
Cloning of acrB gene and site-directed mutagenesis. The acrB gene was amplified from chromosomal E. coli DNA with primers listed in Supplementary  Table 2. The amplified acrB gene was cloned into pET24a (Novagen) via NdeI and XhoI restriction sites 31 . Site-directed mutagenesis was achieved using the ExSite protocol (Stratagene) with 5′-phosphorylated primers (Supplementary Table 2). All the plasmids were verified by sequencing (Eurofins).
Overproduction and purification of DARPins. A single colony of E. coli XL1-Blue cells harbouring pQE30-DARPin 9,32 was used to inoculate LB supplemented with 50 mg ml −1 kanamycin at 37°C and cultivated overnight. Overnight cultures were used to inoculate fresh LB supplemented with antibiotic as above. Gene expression was induced by addition of 0.5 mM (final concentration) isopropyl-β-Dthiogalactoside at OD 600 of 0.7 and the induced cultures were grown overnight at 37°C. Cells were harvested by centrifugation and suspended in 50 mM Tris-HCl buffer at pH 7.5, 400 mM NaCl, and 10 mM Imidazole). Cells were lysed by a Pressure Cell Homogeniser (Stansted Fluid Power Ltd, United Kingdom) at 15,000 psi and cleared by centrifugation at 160,000 × g for 1 h. Supernatant was loaded onto a HisTrap HP Ni 2+ affinity column (5 ml bed volume, GE Healthcare). After two wash steps with the same buffer supplemented with 20 mM and 50 mM of imidazole, DARPin proteins were eluted with 50 mM Tris-HCl buffer at pH 7.5, 400 mM NaCl, 250 mM Imidazole and 10% Glycerol).
Overproduction and purification of AcrB and AcrB mutants. E. coli C43 (DE3) ΔacrAB harbouring pET24acrB His 31,32 (WT or various AcrB variants, respectively) was grown overnight in LB or Terrific Broth supplemented with 50 mg ml −1 kanamycin at 37°C. Overnight cultures were inoculated into fresh LB or Terrific Broth supplemented with antibiotic as above and grown until OD 600 of 0.8 before 0.5 mM (final concentration) isopropyl-β-D-thiogalactoside was added to the culture. Cultures were grown at 20°C for another 16 h and cells subsequently harvested. The cell pellet was resuspended in Buffer A (20 mM Tris-HCl at pH 8.0, 500 mM NaCl, 2 mM MgCl 2 , and 0.2 mM diisopropyl fluorophosphate) and lysed by a Pressure Cell Homogeniser (Stansted, United Kingdom). Cell debris was removed by centrifugation at 23,000 × g for 15 min and cell membranes were collected by centrifugation at 160,000 × g for 2 h. Cell membranes were suspended in Buffer B [20 mM Tris/HCl buffer at pH 7.5, 150 mM NaCl, 20 mM Imidazole, and 10% Glycerol and solubilized with 1% dodecyl maltoside (D-97002-C, DDM, Glycon)] at 4°C for 1 h. Solubilized membranes were cleared at 160,000 × g for 30 min and the supernatant was loaded onto a HisTrap HP Ni 2+ affinity column (1 mL bed volume, GE Healthcare). The column was washed twice with Buffer B supplemented with 0.02% DDM in addition to 60 mM or 90 mM imidazole, respectively. AcrB protein was eluted with Buffer D (20 mM Tris/HCl at pH 7.5, 150 mM NaCl, 220 mM Imidazole, 10% Glycerol, and 0.02% DDM).
Diffraction data collection and refinement. Data were collected on beamline PROXIMA 1 (Pilatus 6 M or Eiger X 16 M detector), PROXIMA 2 A (ADSC Q315r Area or Eiger X 9 M detector), Soleil Synchrotron, Saint Aubin, France, P13 (Pilatus 6 M detector), Petra III, Deutsches Elektronen Synchrotron, Hamburg, Germany or X06DA (Pilatus 6 M detector), Swiss Light Source, Paul Scherrer Insitute, Villigen, Switzerland, indexed and integrated with XDS 33 . The dataset of AcrB-G616P structure in complex with erythromycin and 3-formyrifamycin SV was processed by STARANISO server (Global Phasing). All the structural models were iteratively built in COOT 34 and refined with REFMAC5 35 within CCP4i package 36 , phenix.refine 37 within Phenix package 38 or BUSTER (Global Phasing) 39 . The structure was validated with MolProbity 40 . Polder maps were calculated by phenix.polder 41 within Phenix package 38 . All figures were generated by Pymol (Schrödinger, LLC). Tunnels and cavities were calculated using Caver 42 . Data collection and refinement statistics are listed in Supplementary Data 3.
Thermal shift assay/differential scanning fluorimetry. Differential scanning fluorimetry (DSF) was performed according to Alexandrov et al. 43 with Rotor-Gene Q instrument (Qiagen, Hilden, Germany). The thiol-reactive fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide CPM dye (Sigma, Ex: 380 ± 20 nm; Em: 460 ± 20 nm) was used to study the thermal denaturation of AcrB and variants (0.1 mg ml −1 ) in the presence and absence of the AcrB inhibitor MBX3132 10 (250 μM final concentration). Protein (±inhibitor) sample (59 ul, 0.1 mg ml −1 ) + 1 ul CPM (10 mg/ml) was incubated at RT for 2 min before centrifugation of the sample at 13,000 × g for 10 min at 4°C. Supernatant (25 ul) was transferred into a PCR tube and thermal denaturation scanning was conducted between 25 and 80°C with 1°C/20 s. After collection of data, the fluorescence intensity was plotted against temperature and differential plot (dF (fluorescence)/dT (temperature)) of the curve produced a peak representing protein denaturation state with respect to temperature. The highest point on the peak was defined as the melting temperature (T m in°C) of the protein.
Drug agar plate assay. Drug agar plate assay was performed as previously described 19 . A colony of E. coli BW25113 ΔacrB harboring pET24acrB-His wildtype and AcrB variants were grown overnight in LB containing 50 μg ml −1 kanamycin at 37°C. Dilution of the cultures to OD 600 10 −1 -10 −6 were prepared and 4 μl of each diluted cultures were spotted on an LB agar plate containing 50 μg ml Plates were incubated at 37°C for 14-16 h. Drug agar plates were imaged by ImageQuant (GE Healthcare BioSciences AB, Uppsala, Sweden). The intensity of the cell growth was quantified by ImageJ 1.52o software. The cell growth of WT at first dilution is set to 1. The intensity of cell growth at each dilution step higher than 10% of cell growth of WT at first dilution is considered as cell growth. To calculate the relative cell growth, the cell growth of each mutant is normalized with the cell growth of WT (e.g. if the cell growth of WT is four dilution steps, the fourth dilution step is set to 100% growth). Growth of AcrB variants lower than 75% compared to wild-type AcrB growth is considered significantly affected if p value ≤ 0.005 or slightly affected if p-value = 0.005-0.05. Growth of AcrB variants in between 75 and 85% of growth of wild-type AcrB is considered as slightly affected if p-value ≤ 0.005. p-value was calculated by two-sided Student's t-test.
Agar plate assay supplemented with DDM. A colony of E. coli BW25113 ΔacrB harboring pET24acrB His wild-type or AcrB variants were grown overnight in LB containing 50 μg ml −1 kanamycin at 37°C. Dilution of the cultures to OD 600 10 −3 -10 −8 were prepared and 1.5 μl of each diluted cultures were spotted on an LB agar plate containing 50 μg ml −1 kanamycin, supplemented with 56 mg ml −1 DDM or 12 μg ml −1 doxorubicin. Plates were incubated at 30°C for 14-16 h. Drug agar plates were imaged by ImageQuant TL (GE Healthcare BioSciences AB, Uppsala, Sweden).
Western blot analysis. Western blot analysis of the production of AcrB or AcrB variants was performed as previously described 25 . Briefly, overnight cultures were suspended in 20 μl of Lysis Buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10% Glycerol) to obtain an OD 600 = 15.0, and subsequently, incubated with 2× SDSlysis buffer. The cell suspensions were incubated at 95°C for 10 min. The cell lysates were centrifuged at 13,000 × g for 10 min. The supernatant was resolved by 12.5% SDS-PAGE gels and transferred onto nitrocellullose membrane. The membrane was incubated with anti-AcrB antibody (dilution of 1:10,000; Neosystems, France, custom-antibody) and then, with anti-rabbit IgG (whole molecule)alkaline phosphatase antibody (dilution of 1:1,500; A3687, Sigma-Aldrich, St. Louis, USA). Finally, the blot development was performed with NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt). The wild-type AcrB and all the substitution variants except AcrB-Tyr35Ala, show equal expression/production in E. coli BW25113(DE3) ΔacrB harbouring the complemented gene on a plasmid. Western blot analysis results are given in Source Data Supplementary Data 1 and 2.
Substrate(s) protection cross-linking assay. AcrB cysteine-less variant proteins with amino acid substitution of Ala981Cys (cl_Cys981) or Ser875Cys (cl_Cys875) were purified with the same procedure as shown above until Ni 2+ -affinity chromatography. Finally, cl_Cys981 or cl_Cys875 was purified by size-exclusion chromatography (Superose 6, GE Healthcare) with buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.02% DDM. To determine the efficiency of MTSrhodamine labeling to cl_C981 located at the TMD-BP, approximately 2 μM Subsequently, the reaction was diluted with 100 μl ice-cold PCB in addition of 20 μM MTS-rhodamine (final concentration: 10 μM of MTS-rhodamine in the final mixture) and incubated at room temperature for 5 min. For substrate protection experiment, 2 μM of each AcrB variants, cl_Cys981 or cl_Cys875, were mixed with various concentration of AcrB substrates (erythromycin, fusidic acid, linezolid, novobiocin, oxacillin, and chloramphenicol) in total reaction of 100 μl in PCB buffer and incubated at 22°C for 3 h. Subsequently, 0.5 μM of MTS-rhodamine was added to the reaction and incubated on ice for 10 s. The labelling reaction was stopped by addition of 100 μl ice-cold PCB in the presence of 2 mM NEM (final concentration: 1 mM NEM the reaction mixture). Immediately, all samples were mixed with 2× Laemmli sample buffer in the presence of 2 mM NEM and subjected to SDS-PAGE analysis. The fluorescence signal was detected on an ImageQuant LAS 4000 [Excitation with Epi-Green (Cy3) and emission filter of 575DF20 (Cy3)] (GE Healthcare BioSciences AB, Uppsala, Sweden) before staining with Coomassie Brilliant Blue. All images were analyzed with ImageQuant TL 8.1 software. The relative fluorescence with the signal in the absence of drugs set to 1 and normalized with the quantified Coomassie stained protein fragments (Source Data Fig. 6c, Source Data Supplementary Fig. 4e, f). The apparent inhibition constant of MTSrhodamine labeling for fusidic acid, oxacillin, or novobiocin was calculated by fitting the curves to the nonlinear regression fit with one-site saturation binding function using GraphPad Prism 7.0.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.