Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria^*

Construction of Actinobacillus actinomycetemcomitans MacA-MexAα Hybrid and A. actinomycetemcomitans MacA-OprMα Hybrid Dimer

To construct A. actinomycetemcomitans MacA-MexAα hybrid, DNA fragments encoding A. actinomycetemcomitans MacA residues 29–88, P. aeruginosa MexA residues 95–158, and A. actinomycetemcomitans MacA residues 181–394 were inserted into the NcoI and XhoI sites of the pPROEX-HTA (Invitrogen) vector, resulting in the vector pPRO-AaMacA-MexAα.

To construct the expression vector for A. actinomycetemcomitans MacA-OprMα hybrid dimer, DNA fragments encoding A. actinomycetemcomitans MacA residues 29–123, P. aeruginosa OprM residues 189–212, and A. actinomycetemcomitans MacA residues 148–394 without the stop codon were adjoined using the overlapping PCR technique, and then ligated into the NcoI and XhoI sites of pPROEX-HTA (Invitrogen), resulting in the vector pPRO-AaMacA-OprMα1-nostop. For the second A. actinomycetemcomitans MacA module, DNA fragments encoding A. actinomycetemcomitans MacA residues 29–123, P. aeruginosa OprM residues 396–419, and A. actinomycetemcomitans MacA residues 148–394 (including a stop codon) were adjoined to the XhoI and HindIII sites of the pPROEX-HTA vector. The second A. actinomycetemcomitans MacA module was inserted into the pPRO-AaMacA-OprMα1-nostop vector at the XhoI and HindIII sites, resulting in the vector pPRO-MacA-OprMα hybrid dimer.

Overexpression and Purification of Recombinant Proteins

A. actinomycetemcomitans MacA-MexAα hybrid and A. actinomycetemcomitans MacA-OprMα hybrid dimer proteins were overexpressed in E. coli and purified using the same method used for A. actinomycetemcomitans MacA as described previously (29).

Electron Microscopy and Image Processing

The sample was applied to a freshly plasma cleaned carbon-coated copper grid and allowed to adhere for 60 s before being blotted. Immediately after blotting, 3 μl of a 1% solution of uranyl formate was applied onto the grid and blotted off directly. This was repeated three times. Data were acquired using a Tecnai F20 Twin transmission electron microscope operating at 200 kV, using a dose of ∼40 e⁻/Ų and nominal underfocus ranging from 1.5 to 2.5 μm. At the specimen level, 1,440 high magnification images were automatically collected at a nominal magnification of 80,000× and pixel size 0.107 nm. All images were recorded with a Tietz F415 4k × 4k pixel CCD camera (15-μm pixel) utilizing Leginon data collection software (32). Experimental data were processed by the Appion software package (33), which interfaces with the Leginon database infrastructure. Automatic selection of 44,711 particles was conducted (34) from the micrographs and extracted at a box size of 400 pixels. XMIPP reference-free maximum likelihood alignment was applied to identify the best particles (35) from the selection. The final stack contained 2,244 particles that represented side views of the complex. The initial model was created by applying C6-symmetry on a manually selected two-dimensional average, which was formed using the XMIPP reference-free maximum likelihood alignment procedure (36). The three-dimensional reconstruction was carried out using the XMIPP reconstruction package (36). Resolution was assessed by calculating the Fourier shell correlation at a cut-off of 0.5, which provided a resolution of 24 Å. Automatic Docking was performed using the Chimera software package (37).

Crystallization, Data Collection, and Structure Determination of A. actinomycetemcomitans MacA-MexAα

A. actinomycetemcomitans MacA-MexAα hybrid (20 mg/ml) was crystallized in a precipitation solution containing 0.16 m calcium chloride dehydrate, 0.1 m HEPES sodium (pH 7.5), and 23.5% PEG 400 at 14 °C. The data set was collected on beamline 44 at Spring-8 with the CCD detector MX-225 (Marresearch) at −100 °C. The diffraction data were processed and scaled using the HKL2000 package (38). The resulting crystal belongs to the space group P321 with cell dimensions of a = b = 151.7 Å and c = 216.9 Å. Initial phases were determined using the molecular replacement package MOLREP (39) and the coordinates of a truncated version of the A. actinomycetemcomitans MacA structure at the α-hairpin tip region as a search model. Model building was performed using the program COOT (40), and model refinement was conducted using the programs PHENIX.REFINE (41) and REFMAC5 (39). Crystallographic data statistics are summarized in Table 1. All figures were prepared using PyMOL (42).

Refinement of A. actinomycetemcomitans MacA

The crystallization, data collection, and structural determination of A. actinomycetemcomitans MacA were described previously (29, 43). To build the complete model for A. actinomycetemcomitans MacA, the membrane proximal domain from ZneB was fitted to the MAD-phasing electron density map of A. actinomycetemcomitans MacA. The residues located in the ZneB membrane proximal domain were substituted with those of A. actinomycetemcomitans MacA and subsequently refined using the program PHENIX.REFINE (41).

Size Exclusion Chromatography

To determine the molecular weight of the proteins in this study, size exclusion chromatography was performed at a flow rate of 0.5 ml/min on Superdex S-200 HR 10/30 (GE Healthcare) and equilibrated with 20 mm Tris buffer (pH 8.0) containing 150 mm NaCl.

In Vitro Binding Assay Using CNBr-activated Resin

The TolC α-barrel tip, OprM α-barrel tip, MacA-AcrAα hybrid, and A. actinomycetemcomitans MacA-MexAα hybrid all interacted. The MacA-AcrAα hybrid and A. actinomycetemcomitans MacA-MexAα hybrid-coupled resins were prepared using CNBr-activated resin (50 μg; GE-Healthcare) and MacA-AcrAα hybrid (1 mg) or A. actinomycetemcomitans MacA-MexAα hybrid (1 mg) according to the manufacturer's instructions. MacA-TolCα hybrid dimer (3 mg/ml) and/or MacA-OprMα hybrid dimer (3 mg/ml) were incubated with the coupled resin (20 μl) for 2 h at room temperature. Next, the resin was thoroughly washed with PBS and evaluated using SDS-PAGE, with Coomassie Blue used to stain the protein bands.

Purification of Peptidoglycans

Peptidoglycan from E. coli was purified according to the methods of Leduc et al. (44) and Marcyjaniak et al. (45) with modifications. Briefly, E. coli strain K12 was grown at 37 °C in LB medium and harvested at an OD₆₀₀ of ∼0.9. The cell pellet was suspended in 10 ml of distilled water, mixed with an equal volume of 8% SDS, and incubated for 30 min at 100 °C. After standing at room temperature overnight, the sample was centrifuged at 30 °C for 30 min at 1,800 × g (13,000 rpm) in a high speed centrifuge (Hanil). The gel-type pellet was suspended in 1 ml of water and then washed by four cycles of resuspension and recentrifugation in distilled water to remove traces of SDS. Finally, it was resuspended in 0.5 ml of 10 mm Tris buffer (pH 7.5). The resulting insoluble material was treated with DNase at 37 °C for 30 min, after that with α-amylase at 37 °C for 16 h, and then with pronase at 50 °C for 16 h. The used enzymes were washed from the peptidoglycan solution by boiling and centrifugation. The purified peptidoglycans were finally resuspended in 0.5 ml of 10 mm Tris buffer (pH 7.5).

Crystal Structure of MexA α-Hairpin Domain

The MexA in the crystal tends to form the twisted horseshoe assembly during the crystallization (21, 22). The hexameric MexA could be observed only when it anchors to the membrane and/or binds to OprM (15, 23, 24). To obtain the hexameric form of MexA, we initially attempted to determine the crystal structures of the complex of MexA and OprM. However, it was not straightforward due to the integral membrane protein OprM, and the low affinity between the two proteins in the presence of the detergent that stabilizes the membrane protein. To overcome the problem, we had to construct the forced hexamer of MexA using the chimeric approach reported previously (14, 31). This approach has been proven to be effective in representing the functional structure of α-hairpin domain of AcrA, a MexA homolog, in vivo and in vitro (14).

A chimeric protein containing a MexA α-hairpin domain with A. actinomycetemcomitans MacA was generated as a hexamerization framework because A. actinomycetemcomitans MacA has the highest propensity to form a hexamer in solution as compared with other MFPs (29). The MacA α-hairpin domain was substituted by the corresponding region of MexA and resulted in the chimeric protein (named A. actinomycetemcomitans MacA-MexAα hybrid in this study) that is intended to mimic the hexamerized MexA α-hairpin domains that are capable of binding to OprM (Fig. 1A). Because only α-hairpin domains of MFPs are involved in the binding to the cognate OEPs (17, 30), the MexA α-hairpin domain in the chimeric protein should be suitable for investigating the interaction with OprM.

The chimeric protein A. actinomycetemcomitans MacA-MexAα hybrid was purified and crystallized to analyze the structure of the α-hairpin domain of MexA. The hexagonally shaped crystals were obtained and x-ray diffracted to a resolution of 3.5 Å. To build the complete model for the chimeric protein, we refined the crystal structure of A. actinomycetemcomitans MacA, whose experimental electron density and partial model were described previously (Fig. 1B, right) (29). Because the length of the α-hairpin of MexA is shorter than that of MacA, the end portion of the α-hairpin of A. actinomycetemcomitans MacA was cut to make a searching model for the molecular replacement approach (Fig. 1B, left).

The overall crystal packing of A. actinomycetemcomitans MacA-MexAα hybrid was similar to that of A. actinomycetemcomitans MacA, albeit a different space group (Fig. 1C). The six α-hairpin domains from the six protomers were packed side-by-side to make an α-barrel with a central hollow, as observed in the A. actinomycetemcomitans MacA crystal (Fig. 1D). When compared with α-hairpin domains from the full-length MexA structure (21, 22), any significant conformational change was not observed (supplemental Fig. S1). The end of the α-barrel was slightly flared because the first α-helices of the α-hairpins is twisted outward by ∼60° at the tip region, exposing a six-bladed cogwheel (supplemental Fig. S2). The cogwheels of the chimeric protein intermeshed with the α-barrel from the neighboring molecules in the crystal (Fig. 1C, left). This crystal structure illustrates the propensity for hexameric cylinder formation in the α-hairpin of MexA, suggesting that MexA can form a funnel-like hexamer when the MexA is anchored to the membrane or binds to MexB or OprM.

MexA α-Barrel Domain Interacts with OprM α-Barrel Tip Region

To examine whether or not the MexA α-hairpin domain is capable of interacting with the α-barrel tip region of the cognate OEP OprM, as observed with AcrA and TolC, we constructed a chimeric protein containing the OprM α-barrel tip region based on the hexameric protein A. actinomycetemcomitans MacA, as used in construction of the TolC α-barrel tip region in the previous reports (14, 31). The OprM monomer consists largely of two repeats, which contain one α-hairpin at the α-barrel tip region such as TolC, whereas A. actinomycetemcomitans MacA has only one α-hairpin per monomer. To mimic the α-barrel tip region of OprM, two A. actinomycetemcomitans MacA protomers were fused in tandem and each of the A. actinomycetemcomitans MacA residues 124–147 were replaced with corresponding OprM residues, 188–212 (in repeat 1) or 396–419 (in repeat 2) (Fig. 2A). We designated the chimeric protein as A. actinomycetemcomitans MacA-OprMα hybrid dimer and generated a model based on the crystal structure of A. actinomycetemcomitans MacA (29), whose structure was refined in this study. The overall structure of the model for the chimeric protein was expected to be similar to A. actinomycetemcomitans MacA, to include the feature that the central channel is wide open in the OprM residue tip region (Fig. 2B). This open conformation is distinguished from the closed conformation observed in the crystal structure of the full-length OprM (Fig. 2C) (46).

This chimeric protein containing the OprM α-barrel tip region (A. actinomycetemcomitans MacA-OprMα hybrid dimer) exhibited a strong affinity to another chimeric protein containing MexA α-hairpin on a size exclusion chromatographic column (Fig. 3A), as observed in a study of E. coli AcrA and TolC (14). Interestingly, the E. coli TolC α-barrel tip-containing protein, referred to as A. actinomycetemcomitans MacA-TolCα hybrid dimer in the previous report (14), demonstrated a strong affinity to the MexA α-hairpin-containing protein and vice versa (Fig. 3B). This result indicates that E. coli AcrA and P. aeruginosa MexA share common structural motifs in TolC binding, which is consistent with a previous observation that a chimeric AcrA containing the MexA α-hairpin domain was as functional as the wild type AcrA in vivo (17).

It was reported previously that the stoichiometry of the MexA-OprM complex in a reconstituted membrane bilayer shows strong pH dependence (15). To examine the pH dependence of the MexA-OprM complex formation under detergent-free condition, we carried out the in vitro binding assay. The MacA-OprMα hybrid dimer protein was coupled to agarose resin, and then the MacA-MexAα hybrid protein was incubated at various pH values. After washing, the binding was monitored in Coomassie Blue-stained SDS-polyacrylamide gel (supplemental Fig. S4). The result implied that the binding of the MexA α-hairpin domain to the OprM α-barrel tip is not dependent on the external pH. We think that the protein-protein interaction may be interfered at a certain pH range by the detergent molecules used in a reconstituted membrane layer.

Structure of OprM and MexA Complex, Visualized by Electron Microscopy

To depict the binding interface between OprM and MexA, we carried out electron microscopic studies on the protein complex composed of the two chimeric proteins containing the OprM α-barrel tips and MexA α-hairpins. The hexagonal shape from the top view and indications of bridging from the side view were easily identified in the raw images (Fig. 4A). A projection-matching approach using side views of the negatively stained complex resulted in a three-dimensional density map at 24 Å resolution (Fourier shell correlation = 0.5). Two-dimensional class averages indicate greater flexibility at one of the two ends of the complex (Fig. 4A, arrows). In the resulting three-dimensional electron density maps, this flexibility is represented by additional masses at one of the two ends. In previous reports, similar electron microscopic analyses were done with the TolC α-barrel tip and MacA (31), and the TolC α-barrel tip and AcrA α-hairpin (14).

Compared with previous results (14, 31), the three-dimensional electron density map in this study demonstrated improved detail. The map displayed a characteristic hexagonal dumbbell-like structure with six spikes at both ends. However, the spikes were interlinked at only one end (Fig. 4B). A hexagonal cylindrical shape with a slight bulge was at the middle region of the dumbbell-like structure. A central channel of varying diameter with funnel-like openings on both ends threads through the complex.

Docking of Atomic Models

To gain more structural details of the MexA-OprM complexes, we docked the atomic models to the electron density map. Given the crystal structure of the chimeric protein containing the MexA α-hairpin (A. actinomycetemcomitans MacA-MexAα; Fig. 1C) and the atomic model of the chimeric A. actinomycetemcomitans MacA protein containing the OprM α-barrel tips (A. actinomycetemcomitans MacA-OprMα hybrid dimer; Fig. 2B), we first generated the complex model of the two structures in the intermeshing cogwheel-to-cogwheel manner. The atomic models were well fitted to the density, but only when the two six-bladed cogwheels intermeshed. The configurations of the atomic models were refined using a three-dimensional cross-correlation function (Fig. 5A). The intermeshing α-barrels from the two proteins were fitted to the central hex-cylindrical electron density map, and the slight bulge at the middle region (about one-third from the bottom; Fig. 4, B, panel i) of the HEX cylinder from the bottom in the electron density map perfectly matched the binding interface between OprM and MexA. The bulge region may display the slightly flared ends of each α-barrel of the proteins, and the position of the bulge reflects the length difference of the α-hairpins because MacA α-hairpins are about two times longer than MexA α-hairpins.

The lipoyl and β-barrel domains were well suited to the spreading region in the electron density map, and the membrane proximal domains were strikingly fitted to the six spikes protruding from each end. We were able to unambiguously identify which side contained the OprM tip despite the structural similarity between the two chimeric proteins because only the A. actinomycetemcomitans MacA-OprMα hybrid dimer containing the OprM tip region had the linker at the membrane proximal domains between the two MacA units. As a final step in building the model, the orientation of the side chains in the OprM and MexA α-barrel tips were adjusted by molecular dynamics using CNS (Fig. 5B) (47).

In this complex model of OprM and MexA α-barrel tips, the conserved amino acids, Arg-119, Leu-123, and Ser-130, of MexA contributed to OprM α-barrel tip binding. This observation was consistent with previous reports that demonstrated the role of the OEP in binding and the functional importance of the three conserved residues (13, 14, 29–31). Arg-119 and Leu-123 were located at the end of the first α-helical region of the α-hairpin facing the right side of the cogwheel, whereas Ser-130 was located in the loop between the two α-helices facing the opposite side of the cogwheel (Fig. 5B). The long aliphatic chain of Arg-119 occupied the empty space between the convex tip of the cogwheel in the OprM α-barrel and the concave face of the cogwheel in the MexA α-barrels (Fig. 5C). The guanidinium group of the arginine residue formed a hydrogen bond with the backbone carbonyl group of the α-barrel tip region of OprM. The hydroxyl group of Ser-130 in MexA formed a hydrogen bond with OprM Ser-198 (or Asn-410 in repeat 2). The most striking interaction was mediated by Leu-123, which is most conserved among MFPs. All the OEPs, including TolC, OprM, and CusC, had a conserved motif at the α-barrel tip region, Val/Thr-Gly-Val, which made a characteristic sharp bend and rendered Val/Thr and Val residues facing in the same direction. The Leu-123 residue of MexA, together with Leu-122, created a hydrophobic interaction with Val-201 in repeat 1 (or Val-408 in repeat 2) of OprM, as observed in the hydrophobic interaction between the helices in the coiled-coil structure. These interactions, mediated by the three conserved residues, are all preserved in the complex models of AcrA-TolC and MacA-TolC (Fig. 5E). Additionally, 18 polar interactions were found between MexA and OprM (supplemental Table S2). For instance, Asp-126 of MexA formed a salt bridge with Arg-190 of OprM repeat 1 or Lys-402 of OprM repeat 2.

TolC and AcrA Affinity to Peptidoglycan from E. coli

In Gram-negative bacteria, a peptidoglycan layer transverses the periplasmic space where OEP and MFP interact in vivo. It has been reported that a periplasmic domain of TdeA, a TolC homolog, has an intrinsic affinity to peptidoglycan (48). For these reasons, we examined whether or not the purified proteins of the full-length TolC and AcrA interact with peptidoglycan from E. coli. We used peptidoglycan from the Gram-positive bacteria Staphylococcus aureus as a negative control. The results indicated that TolC and AcrA co-precipitated with peptidoglycan from E. coli but not with that from S. aureus (Fig. 6). These observations indicated that both TolC and AcrA proteins have a specific affinity toward E. coli peptidoglycan. We also observed that the amounts of TolC and AcrA that co-precipitated increased by co-incubation of the two proteins with the E. coli peptidoglycan (Fig. 6). Our findings further suggested that the binding affinity of TolC to AcrA was promoted by peptidoglycan.