Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

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.

Figure 1

Overall structure of MdfA-Cm. The backbone of MdfA is shown in tube representation, with N-domain, C-domain and α_6-7 in green, cyan and orange, respectively. The substrate Cm is shown as magenta stick model. Positions of E26 and D34 are marked as red spheres, and R112 as a blue sphere. The inward-facing cavity is shown as dot-surface representation. TMs are labelled in the right panel.

Figure 2

Structural and functional studies of MdfA in Cm binding. (A) Stereo view of the titratable residues. E26, D34 and their surrounding residues are shown as stick models. Distances between selected atom pairs are marked as dash-lines and labelled. Backbone of the protein is shown in wheat tubes. The substrate Cm is removed for clarity. (B) Stereo view of the Cm binding. The substrate Cm and its surrounding residues are shown as stick models. H-bonds shorter than 3.0 Å are shown as blue dash-lines. Electron density of the omit map is contoured at 3 σ (blue) and 2 σ (wheat) levels. The views of A and B are different by ∼180°. (C) ITC analysis on Cm binding.

Figure 3

Stereo view of motif-B in the 3D structure. Residues of motif-B and surrounding conserved residues are shown as stick models. Selected Gly residues (Cα atoms, wheat coloured) and water molecules (red) are shown in spheres. Backbones of the N- and C-domains are shown in tubes, coloured in wheat and red, respectively.

Figure 4

Drug resistance of MdfA variants. For each variant of MdfA, a serial dilution of cell culture was spotted on solid medium containing 0.2 mM IPGT and 2.5 μg/ml Cm. Photos were taken 36 h after incubation at 37 °C. Experiments were repeated at least three times, and the results were reproducible. Representative results are shown (also see Supplementary information, Figure S7). Positions of the mutation sites are labelled in Figures 2A, 2B and 3.

Figure 5

smFRET data. (A) Cm resistance of WT^* and WT^**. C43 cells expressing the two MdfA variants, Cys-free background (WT^*) and V167C/N374C/WT^* (WT^**), showed similar level of drug resistance to 2.5 μg/ml of Cm as the WT. (B) FRET histograms from single-molecule traces of WT^**, P154A/WT^** and P158A/WT^**. Note that protein samples used in this experiment were incubated in DDM (0.05%), pH 8.0. Data were collected in the absence (left) and presence (right) of Cm (100 μM). Results of two-peak, nonlinear Gaussian fit (with the software Origin) are shown as solid lines in cyan and red, representing the outside-open (C_Out) and outside-close (C_In) states, respectively. The black dotted line is for the summation of the two Gaussian distributions.

Figure 6

Schematic diagram of a mechanistic model depicting the transport cycle of MdfA.