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Structural basis for the inhibition of bacterial multidrug exporters

Nature volume 500, pages 102–106 (2013)Cite this article

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Abstract

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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Figure 1: Crystal structure of the inhibitor-bound AcrB trimer.
Figure 2: Crystal structure of the inhibitor-bound MexB trimer.
Figure 3: Comparison of the close-up views of the inhibitor-binding site of multidrug efflux transporters.
Figure 4: The inhibitory effect of ABI-PP.

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Accession codes

Accessions

Protein Data Bank

Data deposits

The coordinates for ABI-PP-bound AcrB, drug-free MexB, and ABI-PP-bound MexB have been deposited in the Protein Data Bank under accession numbers 3W9H, 3W9I and 3W9J, respectively.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan,

    Ryosuke Nakashima, Keisuke Sakurai & Akihito Yamaguchi

  2. Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan,

    Seiji Yamasaki, Katsuhiko Hayashi & Akihito Yamaguchi

  3. Daiichi Sankyo Co. Ltd, 16-13, Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan,

    Chikahiro Nagata, Kazuki Hoshino & Yoshikuni Onodera

  4. Laboratory of Microbiology and Infectious Diseases, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan,

    Kunihiko Nishino

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Contributions

R.N., K.S., K.Hayashi and C.N. performed the crystallographic analysis. S.Y. and K.N. performed the molecular biological and biochemical analyses. K.Hoshino and Y.O. prepared the inhibitor. A.Y. designed the research and wrote the manuscript.

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Correspondence to Akihito Yamaguchi.

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This file contains a Supplementary Discussion, Supplementary Figures 1-14 and Supplementary Tables 1-2. (PDF 2560 kb)

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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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