Abstract
Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.
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Acknowledgements
We thank the staff of the Protein Research Core Facility at the Institute of Biophysics, Chinese Academy of Sciences (CAS) for their excellent technical support. We are grateful to staff members of SSRF (China), SPring-8 (Japan) and KEK Photon Factory (Japan) synchrotron facilities for their assistance in crystal screening and data collection. We also thank Dr Zhenfeng Liu for the help with data collection, and Dr Juelich T for linguistic assistance during the preparation of this manuscript. This work was supported by the National Basic Research Program of China (973 Program; 2011CB910301 and 2014CB910104 to XCZ, and 2014CB910400 to YFZ), the Chinese Academy of Sciences (XDB08020301 to XCZ) and the National Natural Science Foundation of China (31470745 to XCZ, and 31200560 to XW).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Analysis of amino acid sequences of MdfA (PDF 746 kb)
Supplementary information, Figure S2
The WebLogo representation of MFS transporters. (PDF 107 kb)
Supplementary information, Figure S3
Structural comparison between MdfA and YajR. (PDF 185 kb)
Supplementary information, Figure S4
Substrate binding in MdfA (PDF 119 kb)
Supplementary information, Figure S5
Actual ligands in our crystal structures in comparison to related known substrates. (PDF 40 kb)
Supplementary information, Figure S6
Binding of deoxycholate with MdfA (PDF 59 kb)
Supplementary information, Figure S7
Chloramphenicol-resistance assay. (PDF 183 kb)
Supplementary information, Figure S8
Stereo view of distribution of residues discussed in the main text. (PDF 62 kb)
Supplementary information, Table S1
Statistics of diffraction data and structure refinement (PDF 81 kb)
Supplementary information, Table S2
Formation of ligand binding sites. (PDF 39 kb)
Supplementary information, Table S3
Bulk dye anisotropy measurements. (PDF 71 kb)
Supplementary information, Data S1
Supplementary methods (PDF 96 kb)
Supplementary information, Data S2
Additional results and discussion (PDF 73 kb)
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Heng, J., Zhao, Y., Liu, M. et al. Substrate-bound structure of the E. coli multidrug resistance transporter MdfA. Cell Res 25, 1060–1073 (2015). https://doi.org/10.1038/cr.2015.94
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DOI: https://doi.org/10.1038/cr.2015.94
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