Crystal structure of MexB bound with Lauryl maltose neopentyl glycol (LMNG)

Unlike the main RND transporter from Escherichia coli AcrB, bound drugs have not been identified in MexB crystal structures. Regardless of MexB crystalized with or without drugs, the DBP of MexB was occupied by a detergent molecule DDM13,17 (Fig. 1a). In order to identify bound drugs, we exchanged DDM by the large molecular weight detergent LMNG (Fig. 1b), which has a molecular weight almost twice as much as DDM. The molecular weight is not only higher than that of LMMDs (e.g. minocycline and doxorubicin), but is also higher than that of HMMDs (e.g. rifampicin and erythromycin). We expected that LMNG may not bind to DBP and may not disturb drug binding to the DBP.

Figure 1 Chemical structures of n-dodecyl-β-D-maltopyranoside (DDM), lauryl maltose neopentyl glycol (LMNG), CYMAL-7 neopentyl glycol (C7NG), and CYMAL-6 neopentyl glycol (C6NG) used in this experiment. Full size image

Surprisingly, we clearly detected the electron density of bound LMNG in the DBP of the MexB crystal structure, regardless whether or not drug was included in the crystallization medium. Figure 2a,b show the LMNG-bound structure of MexB. The binding site is composed of the PN1, PN2 and PC1 subdomains7 of the binding monomer of MexB (Fig. 2a,b). The hydroxymethyl group and the eight hydroxy groups of the glucose moieties of LMNG form hydrogen bonds with the side chains of Gln46, Glu81, Thr89, Arg128, Lys134, Ser180, Gln273 and Arg620 (Fig. 2d). The binding site of LMNG partially overlaps with the ABI-PP and DDM binding sites (Fig. 2c). One of the alkyl chains of LMNG is inserted into the inhibitor-binding hydrophobic pit13 (Fig. 2c). The strong interaction between bound ABI-PP and the wall of the hydrophobic pit is considered to be the cause of its inhibitory action13. The other alkyl chain of LMNG is elongated parallel in the space above the inhibitor-binding pit. Two maltose moieties are oppositely elongated towards the exit and entrance of the DBP (Supplementary Fig. S1). As a result, the LMNG molecule spreads throughout the DBP space, indicating that the DBP has sufficient space to accommodate a molecule with a molecular mass of more than 1,000, if it can specifically fit to the binding site. Although LMNG has a high molecular mass, the chemical structure is flexible, because of its many rotatable bonds. This highly flexible feature may also contribute to the ability of LMNG to traverse the narrow channel in order to reach the DBP. In addition, principal moments of inertia (PMI) analysis34 of RND pump substrates were plotted (Supplementary Fig. S2). This analysis can visualize structural diversity by categorizing a molecular shape into distinct topologies: rod-, sphere- and disc-like character. It shows that DBP-binding drugs share rod-like features. LMNG is a compound which is able to form a rod-like structure. On the other hand, the PBP-binding substrates do not have this feature. The peristaltic motion, including the swinging of the switch-loop may be necessary to translocate the PBP-binding drugs into DBP and, once they enter into the DBP, do not flow back and are consequently occluded in the DBP13.

Figure 2 Crystal structure of LMNG-bound MexB. (a) Whole trimer structure of MexB (ribbon model) bound with LMNG (electron density map). Binding, extrusion and access protomers are shown in blue, pink and green, respectively. The LMNG-binding site is shown by the electron density calculated as an Fo-Fc omit map contoured at 2.8 σ (orange mesh). (b) Close-up view of the LMNG-binding site. Electron density of LMNG (orange mesh) overlapped with stick models of LMNG (pink). (c) Overlapping view of the bound LMNG (pink), DDM (green, PDB ID: 3W9I) and ABI-PP (yellow, PDB ID: 3W9J) shown in the cut view of the surface model of the distal pocket in the binding monomer. The inhibitor binding hydrophobic pit is shown by red surface. (d) The 2D representation of the interaction between LMNG and MexB (PDB ID: 6IIA) was drawn using LigPlot+40. Full size image

LMNG as a substrate of MexB and a competitive inhibitor of MexAB-OprM-mediated drug efflux

LMNG does not affect the growth of P. aeruginosa (data not shown) probably due to the low permeability of the outer membrane of P. aeruginosa. To reveal the properties of LMNG as a substrate of MexB, acrB-deficient Salmonella enterica serovar typhimurium was transduced with mexAB-oprM-encoding plasmids. Figure 3 shows the effect of LMNG on the growth of S. enterica. The growth of the acrB-deficient S. enterica cells was not affected by LMNG (Fig. 3a). However, the growth of the acrB-deficient rough mutant (ΔacrBΔrfaC) cells35 was significantly inhibited by LMNG in a concentration-dependent manner (Fig. 3b), indicating that the polysaccharide chains of LPS in the outer membrane hinder the penetration and antibiotic action of the LMNG molecules. When MexAB-OprM was expressed, the growth of the ΔacrBΔrfaC mutant was no longer affected by LMNG (Fig. 3c), clearly indicating that LMNG is exported by MexB. The small initial decrease in growth by the addition of 8 μg/mL LMNG (Fig. 3c) is probably due to the effect of disruption of the outer membrane surface by the surfactant action of LMNG. Because the CMC value of LMNG is 10 μg/mL (in water) according to the product description, the degree of the surfactant action was almost saturated at 8 μg/mL. Phe178 is the residue located at the middle of the inhibitor-binding pit and is important for inhibitor-binding, while the F178W mutant (which partly closes the pit and prevents the binding of inhibitor ABI-PP in AcrB) still retains drug export activity13. In this experiment, MexA-MexB(F178W)-OprM was also expressed in S. enterica. The expression level of this mutant was the same as that of the wild type (Supplementary Fig. S3). The growth of MexB(F178W)-expressing cells was not significantly affected by LMNG (Fig. 3d), indicating that the mutant retains its LMNG-export activity. The very slight LMNG-dose-dependent decrease in growth of MexB(F178W) may be caused by a decrease in the export activity by the mutation itself.

Figure 3 Antimicrobial activity of LMNG measured by the effect on growth curves of Salmonella enterica serovar typhimurium. (a) ΔacrB (NKS148). (b) ΔacrBΔrfaC transduced with the vector (pMMB67HE) (NKS1421). (c) ΔacrBΔrfaC expressing MexAB-OprM (NKS1422). (d) ΔacrBΔrfaC expressing MexAB(F178W)-OprM (NKS1423). Full size image

Figure 4 shows the competitive inhibition by LMNG against erythromycin (EM) and ethidium bromide (EtBr) MexB-mediated export. In the presence of 2 μg/mL EM, the ΔacrBΔrfaC cells did not show any growth regardless of the presence or absence of LMNG (Fig. 4a, left panel), while the MexAB-OprM-expressing cells were able to grow in the presence of 2 μg/mL EM. The growth was significantly decreased by the addition of LMNG in a dose-dependent manner (Fig. 4a, middle panel), indicating that LMNG competitively inhibits EM extrusion by MexB as these same concentrations of LMNG alone did not affect the growth at all (Fig. 3c). As for the MexB(F178W)-expressing cells, the presence of 2 μg/mL EM did not inhibit the growth of the cells, however, the overall growth was lower than that of the wild-type expressing cells due to the low EM extrusion activity of the mutant MexB. Nevertheless, contrary to the wild-type expressing cells, the growth of the mutant-expressing cells was essentially not inhibited by LMNG (Fig. 4a, right panel).

Figure 4 Inhibitory effect of LMNG on MexAB-OprM-mediated drug efflux in the rough mutant of acrB-deficient S. enterica. (a) The effect of LMNG on the growth of cells in the presence of a low concentration (2 μg/ml) of erythromycin. Left panel: the strain transduced with the vector, middle panel: the strain expressing MexA-MexB-OprM, right panel: the strain expressing MexA-MexB(F178W)-OprM. (b) The effect of LMNG on the MexAB-OprM-mediated prevention of ethidium bromide accumulation in the ΔacrBΔrfaC mutant of S. enterica. The strain transduced with the vector (red), the strain expressing MexA-MexB-OprM (green) and the strain expressing MexA-MexB(F178W)-OprM (blue) were used. Then, 10 µM ethidium bromide and the indicated amount of LMNG were added. Full size image

Next, we tested the competitive inhibition of LMNG against EtBr export. EtBr accumulates in the ΔacrBΔrfaC cells of the S. enterica strain and yields fluorescence by intercalation into the chromosomal DNA (Fig. 4b, red lines). The EtBr accumulation in the absence of the MDR pump was almost not affected by the addition of LMNG. In the case of the wild-type MexAB-OprM-expressing cells, EtBr accumulation was very low as the drug export activity of MexB prevented EtBr from entering the cells (Fig. 4b, pale green line). When LMNG was added, the prevention became weaker, and the degree of EtBr accumulation increased depending on the LMNG concentration (Fig. 4b, green lines), indicating that LMNG competitively inhibits MexB-mediated EtBr export. The MexB(F178W)-expressing cells showed an intermediate level of EtBr accumulation, between that of the ΔacrBΔrfaC cells and the wild-type MexB-expressing cells, due to the lower but significant EtBr-export activity of MexB(F178W) (Fig. 4b, pale blue line). Contrary to the wild type, the EtBr export activity of the mutant was not affected by LMNG addition at all (Fig. 4b, blue lines). In summary, both EM and EtBr efflux are competitively inhibited by LMNG in wild-type MexB but not inhibited in MexB(F178W).

We obtained similar results with clarithromycin (CAM), ciprofloxacin (CPFX), and azithromycin (AZM) in a growth curve assay (Supplementary Fig. S4a) and also berberine (BER) in a fluorescence accumulation assay (Supplementary Fig. S4b). The export of these drugs by MexB was inhibited by LMNG in wild-type MexB, but not in the F178W mutant. On the other hand, the export activity of doxorubicin (DXR), minocycline (MINO) and rhodamine 6 G (R6G) was not significantly affected by LMNG in both wild-type MexB and mutant MexB (Supplementary Fig. S5). These drugs not affected by LMNG are the drugs of which the DBP-binding structures of AcrB were previously reported14,19. While the affinity of LMNG to MexB could not be measured directly, it should be estimated to be low when considering the concentrations required for the inhibition of the EM export (Fig. 4a, middle panel). Thus, it might not inhibit the drugs clearly binding to the DBP. The drugs inhibited by LMNG in wild type MexB, are drugs that bind to the PBP in AcrB or drugs of which binding structures were not determined in AcrB nor MexB. The binding affinity of these drugs to the DBP should be very low. LMNG might only inhibit the export of DBP-not-bound or DBP-very-low-affinity-bound drugs.

Crystal structure of MexB(F178W) co-crystalized with LMNG

We determined the crystal structure of MexB(F178W) co-crystalized with LMNG (Supplementary Fig. S6). Supplementary Fig. S6a shows a close-up view of the DBP in the MexB(F178W) co-crystal with LMNG. The angle and range of view are the same as those in Fig. 2b. Although slight positive electron densities were observed in the Fo-Fc omit map, it is not sufficient to identify bound LMNG. Supplementary Fig. S6b shows the LMNG molecule in the DBP, which was obtained by docking simulations using Glide (Schrödinger). The alkyl chain of LMNG, which was inserted into the inhibitor-binding pit in the wild-type MexB, was located outside the pit of MexB(F178W). The indole ring of the bulky tryptophan side chain was slightly protruded into the pit (Supplementary Fig. S6b) and likely interferes the insertion of the alkyl moiety of LMNG. In the ABI-PP-bound structure of MexB(F178W)15 the π-π interaction between the indole and pyridopyrimidine ring pushed the indole ring into the flat wall. However, the alkyl chain of LMNG was no longer able to overcome the steric hindrance by the indole ring and could not insert into the pit.

Co-crystal structures of MexB with other high-molecular-mass neopentyl glycol (NG) derivatives CYMAL-7 neopentyl glycol (C7NG) and CYMAL-6 neopentyl glycol (C6NG)

To reveal whether it is a general rule or not that high-molecular-mass NG derivatives can bind in the DBP, we tried to determine the crystal structures of MexB with C7NG and C6NG. These molecules are analogues of LMNG, but the alkyl chains are terminally substituted with cyclohexane (Fig. 1c,d). Figure 5a shows the structure of C7NG-bound MexB. The electron density derived from C7NG was observed in the DBP (Supplementary Fig. S7). While the electron density was poor, one of the alkyl chains was inserted into the pit. The positions of the C7NG and the LMNG molecule almost completely overlapped in the binding site. On the other hand, we could not identify a bound C6NG molecule.

Figure 5 Crystal structure of the C7NG-binding site of MexB (a) and the effect of C6NG (b) and C7NG (c) on the growth of the ΔacrBΔrfaC mutant of S. enterica. (a) Close-up view of the C7NG-binding site in MexB overlapped with bound LMNG. (b,c) The left panels indicate the strain transduced with the vector, the middle panels indicate the strain expressing MexA-MexB-OprM, and the right panels indicate the strain expressing MexA-MexB(F178W)-OprM. Full size image

We measured the effect of C6NG and C7NG on the growth of the ΔacrBΔrfaC S. enterica cells (Fig. 5b,c). C6NG showed growth inhibition against the strain without MDR pumps (Fig. 5b, left panel), but did not affect the growth of wild type MexB-expressing cells (Fig. 5b, middle panel) nor the F178W mutant-expressing cells (Fig. 5b right panel). The initial decrease in the growth at the minimum concentration of C6NG (2 μg/mL) is probably due to the surfactant action of C6NG causing outer membrane disruption, which is independent of the export of C6NG. Thus, similar to LMNG, C6NG is shown to be a substrate of MexB and MexB(F178W). On the other hand, the inhibitory activity of C7NG on the growth of ΔacrBΔrfaC cells was very small (Fig. 5c, left panel). This result may be due to the low permeability of C7NG through the outer membrane, even in the rough mutants. Although there is no biochemical evidence which shows that C7NG is a substrate of MexB, because it does not obstruct growth of host bacteria and does not compete with the efflux activity of MexB (Fig. 5c), we argue that C7NG is a substrate of MexB from the fact that the C7NG binding crystal structure was obtained, and the fact that its NG-colleagues LMNG and C6NG are both substrates of MexB.

Finally, we measured the inhibitory effect of C6NG and C7NG on MexB-mediated EtBr export (Supplementary Fig. S8) by the method described in Fig. 4b. Although there are variations among experiments, the EtBr accumulation-prevention activity of the wild-type MexB is significantly inhibited by C6NG, while that of MexB(F178W) is not significantly inhibited, similar to LMNG. On the other hand, the inhibitory activity of C7NG was not observed, probably reflecting the low permeability of C7NG.