AcrB and its homologues are the principal multidrug transporters in Gram-negative bacteria1,2,3,4,5,6 and are important in antibiotic drug tolerance7,8. AcrB is a homotrimer that acts as a tripartite complex9,10 with the outer membrane channel TolC11,12 and the membrane fusion protein AcrA13,14. Minocycline and doxorubicin have been shown to bind to the phenylalanine cluster region of the binding monomer15. Here we report the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin. These drugs bind to the access monomer, and the binding sites are located in the proximal multisite binding pocket, which is separated from the phenylalanine cluster region (distal pocket) by the Phe-617 loop. Our structures indicate that there are two discrete multisite binding pockets along the intramolecular channel. High-molecular-mass drugs first bind to the proximal pocket in the access state and are then forced into the distal pocket in the binding state by a peristaltic mechanism involving subdomain movements that include a shift of the Phe-617 loop. By contrast, low-molecular-mass drugs, such as minocycline and doxorubicin, travel through the proximal pocket without specific binding and immediately bind to the distal pocket. The presence of two discrete, high-volume multisite binding pockets contributes to the remarkably broad substrate recognition of AcrB.
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Protein Data Bank
The coordinates for unliganded AcrB and the AcrB–rifampicin, AcrB–erythromycin and AcrB–rifampicin–minocycline complexes have been deposited in the Protein Data Bank under accession numbers 3AOA, 3AOB, 3AOC, and 3AOD, respectively.
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We thank T. Tsukihara for advice on the crystallographic analysis. We also thank N. Kato for discussion about the organic chemistry of the drugs that were investigated in this study. Our diffraction data were collected using Osaka University’s beamline BL44XU at SPring-8, which was equipped with an MX225-HE detector (Rayonix) and was financially supported by the Academia Sinica and the National Synchrotron Radiation Research Center (Taiwan). We are also grateful to the technical staff of the Comprehensive Analysis Center of the Institute of Scientific and Industrial Research for their assistance. This work was supported by the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
The authors declare no competing financial interests.
The file contains Supplementary Figures 1-13 with legends and a Supplementary Discussion and additional references. (PDF 2173 kb)
The movie shows conformational change of the Phe-617 loop during functional rotation of AcrB. The movie was generated by morphing the three functional states of AcrB bound to both rifampicin and minocycline using PyMOL. The Phe-617 loop is shown in red. (MOV 3027 kb)
The movie shows movement of the amino acid side chains that interact with erythromycin during drug transport. The side chains of Phe 617, Ser 134 and Ser 135 are shown in red. The movie was generated by morphing the three functional states of the erythromycin-bound form of AcrB using PyMOL. (MOV 1873 kb)
The movie shows crystal structure of AcrB simultaneously bound to rifampicin and minocycline solved at a 3.3 Å resolution. Rifampicin (magenta) in the access monomer (green) and minocycline (cyan) in the binding monomer (blue) are shown in a CPK (Corey-Pauling-Koltun) representation. (MOV 3426 kb)
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Nakashima, R., Sakurai, K., Yamasaki, S. et al. Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket. Nature 480, 565–569 (2011). https://doi.org/10.1038/nature10641
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