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Crystal structures of a multidrug transporter reveal a functionally rotating mechanism

Abstract

AcrB is a principal multidrug efflux transporter in Escherichia coli that cooperates with an outer-membrane channel, TolC, and a membrane-fusion protein, AcrA. Here we describe crystal structures of AcrB with and without substrates. The AcrB–drug complex consists of three protomers, each of which has a different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding. The structures indicate that drugs are exported by a three-step functionally rotating mechanism in which substrates undergo ordered binding change.

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Figure 1: Structure of the AcrB–minocycline complex based on the asymmetric crystal.
Figure 2: Multidrug recognition by AcrB.
Figure 3: The novel drug translocation pathway for AcrB.
Figure 4: Structure with a slab (23 Å) of the transmembrane domain viewed from the periplasmic side.
Figure 5: Schematic illustration of the proposed ‘functionally rotating ordered multidrug binding change mechanism’ mediated by AcrB.

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Acknowledgements

We thank P. J. F. Henderson, C. Toyoshima and T. Tsukihara for discussions, advice and critical reading. We are also indebted to E. Kajitani and N. Kato for synthesis of brominated substrate. Thanks are also due to N. Shimizu, H. Sakai, M. Kawamoto, M. Yamamoto, M. Yoshimura and A. Nakagawa for data collection at SPring-8. Synchrotron experiments were performed on BL41XU and BL44XU in SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute, and the Joint Research Committee of the Institute for Protein Research, Osaka University, respectively. This work was supported by PRESTO and CREST from the Japan Science and Technology Agency and grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Correspondence to Satoshi Murakami.

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Coordinates for the unliganded, AcrB–minocycline complex and AcrB–doxorubicin complex structures have been deposited in the Protein Data Bank under accession numbers 2DHH, 2DRD and 2DR6, respectively. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figures 1–3

This file contains Supplementary Figures 1–3 with accompanying text. This file also contains the Supplementary Movie Legends. (PDF 4397 kb)

Supplementary Figure 4

AcrB–drug complex formation (JPG 69 kb)

Supplementary Tables.

This file contains Supplementary Table 1 for crystallographic statistics and Supplementary Table 2. (PDF 133 kb)

Supplementary Movie 1

Crystal structure of the AcrB-Minocycline complex at 2.8 angstroms resolution. (MOV 4806 kb)

Supplementary Movie 2

A Movie showing the conformation changes in the porter domain of AcrB on the basis of functionally rotating mechanism (MOV 99 kb)

Supplementary Movie 3

Animation movie showing the functionally rotating mechanism of AcrB. (MOV 1282 kb)

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Murakami, S., Nakashima, R., Yamashita, E. et al. Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443, 173–179 (2006). https://doi.org/10.1038/nature05076

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