Crystal structure of AcrB complexed with linezolid at 3.5 Å resolution
AcrB is an inner membrane resistance-nodulation-cell division efflux pump and is part of the AcrAB–TolC tripartite efflux system. We have determined the crystal structure of AcrB with bound Linezolid at a resolution of 3.5 Å. The structure shows that Linezolid binds to the A385/F386 loops of the symmetric trimer of AcrB. A conformational change of a loop in the bottom of the periplasmic cleft is also observed.
KeywordsMultidrug resistance AcrB RND efflux pumps Linezolid Membrane protein Protein–drug complex X-ray crystal structure
Acriflavine resistance protein A
Acriflavine resistance protein B
US food and drug administration
Minimal inhibitory concentration
Protein data bank
Resistance nodulation division
AcrB, the principal multidrug transporter in E. coli, crosses the cytoplasmic membrane and acts as a proton/drug antiporter. It is part of the AcrAB–TolC tripartite efflux system consisting of an outer membrane factor, TolC, a periplasmic membrane fusion protein, AcrA, and the inner membrane resistance-nodulation-cell division (RND) efflux pump, AcrB. This integrated three-component molecular complex extrudes a large variety of cytotoxic substances such as antibiotics, organic solvents, dyes, and detergents from the cell directly into the medium, bypassing the periplasm and the outer membrane [1, 2, 3]. Over-expression of the tripartite RND efflux systems is thought to be a major factor in multidrug resistance (MDR) in Gram-negative bacteria. Effective control of this and related MDR systems has become an emerging focus for global public health efforts [2, 4].
AcrB is one of the most well-studied RND efflux pumps. Crystal structures of AcrB by itself as well as several drug-bound complexes have been structurally characterized [5, 6, 7, 8, 9, 10, 11, 12]. Two types of quaternary arrangement have been observed in structures of AcrB: (1) a symmetric trimer, formed by three identical protomers or protomer–drug complexes; and (2) an asymmetric trimer, consisting of three distinct protomer conformations corresponding to three functional states of the transport cycle: access, binding and extrusion, with drug molecules only bound to the binding protomer. These structures have exemplified several drug-binding modes of AcrB and provided structural insights to the substrate transport mechanism of RND multidrug efflux transporters.
In an effort to explore how AcrB interacts with drugs, we have determined the crystal structure of AcrB complexed with Linezolid, an oxazolidinone-type antibacterial agent that inhibits bacterial protein synthesis by specifically binding to the 50S ribosomal subunit. Linezolid was the first FDA-approved oxazolidinone antibiotic used for the treatment of serious infections caused by Gram-positive bacteria that are resistant to other antibiotics. Therefore, this drug has been called a “reserve antibiotic”, a drug of last resort against potentially intractable infections . Linezolid is a synthetic compound, and is therefore not susceptible to the same mechanisms underlying bacterial resistance against naturally occurring antibiotics. However, it has no clinically significant effect on most Gram-negative bacteria. This is thought to be a result of relatively low intracellular concentration of Linezolid due to efflux . The intracellular concentration of Linezolid could be increased substantially by inhibition of RND-type efflux pumps . E. coli with inactivated AcrB has been found to be more susceptible to Linezolid than cells with an intact pump . Further, NMP (1-(1-naphthylmethyl)-piperazine, a putative efflux pump inhibitor) has been shown to reduce the MIC (minimal inhibitory concentration) of Linezolid by fourfold for E. coli, Citrobacter freundii, Enterobacter aerogenes and Acinetobacter baumannii [16, 17]. Although these data suggest that Linezolid can be extruded by efflux pumps, there is no direct evidence yet to support this hypothesis. Here, we report the crystal structure of AcrB and Linezolid complex, in which AcrB indeed binds Linezolid in the same fashion as several other antibiotics that are extruded by efflux pumps.
Materials and methods
Cloning, overexpression, and purification
Wild-type AcrB with a C-terminal polyhistidine tag was prepared as described previously . Briefly, AcrB was overproduced in E. coli JM109 with a histidine-tagged AcrB-overexpersion plasmid pAcBH. The cells were disrupted with Microfluidizer (Microfluidics Corp.) and the membrane fractions were collected washed using several ultracentrifugation steps at 150,000g for 90 min. Purified membranes were solubilized in buffer containing 50 mM Tris–HCl, pH 7.0, 10 % glycerol in 2 % n-dodecyl-β-d-maltoside (DDM) (Anatrace). Lipids and debris were removed by ultracentrifugation at 170,000g for 60 min. Extracted histidine-tagged AcrB was purified with metal affinity column chromatography equilibrated with buffer (20 mM Tris–HCl, pH 7.5, 0.3 M NaCl, 10 % glycerol and 0.2 % DDM). The column was washed stepwise using 25 and 100 mM imidazole added to the above buffer. Purified AcrB was eluted with 300 mM imidazole. Imidazole was then removed by three concentration-dilution steps using an ultrafiltration membrane. Proteins were concentrated to 28 mg/mL in 20 mM sodium phosphate (pH 6.2), 10 % (v/v) glycerol and 0.2 % (w/v) DDM and were frozen in liquid nitrogen.
Crystallization and data collection
AcrB was crystallized using the sitting-drop vapor diffusion method with 0.1 M NaCl, Na-phosphate pH 6.2, and 8 % PEG 4000 as crystallization reagents. Crystals of the AcrB–Linezolid complex were obtained by soaking the AcrB crystals in a solution containing Linezolid prior to data collection. Linezolid stock solution (30 mM) was prepared with water, and 6 mM Linezolid was added to a cryosolvent containing the crystallization reagents plus 25 % glycerol. Apo-AcrB crystals were transferred to the cryosolvent and incubated at 21 °C for 10 min before flash cooling in liquid nitrogen. X-ray diffraction data were collected at 100 K on the 5.0.2 beam line at the Advanced Light Source at the Lawrence Berkeley National Laboratory with X-rays at a wavelength of 1 Å. The crystal diffracted better than 3.3 Å initially and decayed during data collection, leading to a useful resolution of about 3.5 Å by the end of data collection. The diffraction data were processed with the HKL2000 program suite . The AcrB–Linezolid complex belongs to the space group R32 with cell parameters a = b = 144.7 Å, c = 519.4 Å. The solvent content is 74 % assuming 1 molecule in the asymmetric unit.
Structure determination and refinement
X-ray data and refinement statistics
a, b, c (Å)
144.7, 144.7, 519.4
α, β, γ (°)
90, 90, 120
Matthews coefficient (Å3/Da)
Solvent content (%)
No. of unique reflections
No. of reflections in Rfree set
Overall completeness (%)
RMSD bond lengths (Å)
RMSD bond angles (°)
MolProbity Ramachandran distribution
Most favored (%)
Mean main chain B-factor (Å2)
Mean overall B-factor (Å2)
Mean solvent B-factor (Å2)
Protomers in ASU
A (7-498, 513-864, 869-1,036)
No. of protein atoms
No. of ligand atoms
No. of water molecules
PDB accession code
Structure validation and deposit
The quality of the final structure was assessed using MolProbity  and phenix.refine. The atomic coordinates and structure factors are available in the Protein Data Bank under accession code 4K7Q.
Results and discussion
Overall structure of AcrB–linezolid complex
Comparison with apo-AcrB structure
The overall structure of AcrB–Linezolid complex was very similar to that of AcrB by itself . There was a significant local conformational difference at residues 670–675. When superposed together, the two structures has a root-mean-square Cα coordinate deviation of 0.4 Å while these residues, on the surface of the protein but not near crystal contacts, differ by up to 4 Å in backbone position. These residues reside in a loop lining the bottom of the cleft in the porter domain thought to be important for substrate transport and AcrA binding. It is possible that the change in position of this loop between unbound and Linezolid-bound states may reflect a functionally important state of AcrB, though we cannot rule out the possibility that the shift in the 670–675 loop was due to slight differences between crystals used for structure determination. We are in the process of obtaining higher resolution data of the AcrB–Linezolid complex as well as AcrB by itself under identical crystallization and data collection conditions.
The crystal structure of an AcrB–Linezolid complex has been determined at a resolution of 3.5 Å. The structure shows that one Linezolid binds to the A385/F386 loop of each protomer in the symmetric trimer. This loop has previously been shown to interact with Ethidium, Nafcillin, and Ampicillin. A conformational change is also found in a loop at the bottom of the periplasmic cleft thought to be important for AcrA binding and drug transport.
The authors thank the Los Alamos National Laboratory Directed Research and Development Program for a Feasibility Studies Program for Study grant for support of this work. This work is partially supported by the funding program for Next Generation World-Leading Researchers (NEXT Program) and the program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) and by the ERATO “Lipid Active Structure Project” from Japan Science and Technology Agency (JST). We would like to thank the staff at the beam lines 5.0.1 and 5.0.2 managed by the Berkeley Center for Structural Biology (BCSB) at the Advanced Light Source (ALS) for technical support. The BCSB is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
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