The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens1,2. However, despite efforts to develop efflux inhibitors3, clinically useful inhibitors are not available at present4,5. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY6,7; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa8,9,10. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics11,12,13. Drugs were shown to be exported by a functionally rotating mechanism12 through tandem proximal and distal multisite drug-binding pockets13. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π–π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.
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We thank T. Nakae for providing the plasmids encoding mexB, mexY, mexAB-oprM and mexXY-oprM. We also thank N. Kato for discussion regarding the organic chemistry of the inhibitor that was investigated in this study. Our diffraction data were collected using Osaka University’s BL44XU beamline at SPring-8, which was equipped with an MX225-HE CCD detector (Rayonix) and was financially supported by the Academia Sinica and the National Synchrotron Radiation Research Center (Taiwan). We thank K. Harada for assistance with the liquid chromatography–tandem mass spectrometry assay. This work was supported by CREST from the Japan Science and Technology Agency, 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.
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Nakashima, R., Sakurai, K., Yamasaki, S. et al. Structural basis for the inhibition of bacterial multidrug exporters. Nature 500, 102–106 (2013). https://doi.org/10.1038/nature12300
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