An embedded lipid in the multidrug transporter LmrP suggests a mechanism for polyspecificity

Abstract

Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.

Figure 1.. DEER spectroscopy shows limited conformational change upon substrate binding to LmrP.

The conformational equilibrium of LmrP is compatible with the outward facing conformation in the apo state (blue) and in the presence of Hoechst 33342 (green), ethidium bromide (red), roxithromycin (cyan), TPP⁺ (yellow), verapamil (grey) and tetracycline (magenta), as evidenced by distance distributions observed with spin-labels located on the extracellular (EC) and intracellular (IC) termini of transmembrane helices. Distributions were normalised. Interspin distance is denoted by r, with P(r) indicating the distance probability. The * denotes the peak due to aggregation in the sample (see methods).

Figure 2.. The structure of LmrP in complex with Hoechst 33342.

a) Cartoon representation of LmrP with N-lobe, central loop and C-lobe coloured in brown, dark red and light brown respectively. Meshes represent F_o-Fc maps at 2 σ after molecular replacement and refinement without modelled ligand. b) Hoechst 33342 (green) can be modelled (with the resultant 2F_o-F_c map following refinement shown at 1 σ in green) and forms polar interactions with D235 and E327, located in the C-lobe of LmrP.

Figure 3.. A lipid within the binding cavity of LmrP stabilises the observed structure.

a) Phosphatidic acid, shown as cyan sticks, can be modelled into the unaccounted density observed in the binding cavity of LmrP, shown as a blue mesh of the 2F_o-F_c map at 1 σ. b) Across microsecond simulations of LmrP bound to Hoechst 33342 and with POPG within the binding pocket, the 3D mass-density map of POPG (teal mesh) adopts a similar conformation to that of the electron density of the lipid observed in the crystal structure. c) The polar residues R14 and D142 within the binding pocket of LmrP adopt a similar position to that observed in the crystal structure during simulations in the presence of Hoechst 33342 and POPG, but re-orient in simulations without POPG, and R14 is ultimately exposed to the intracellular space. The side-chains of R14 and D142 observed in the crystal structure are shown in grey, with 3D mass-density maps superposed from the simulation with both Hoechst 33342 and POPG (teal), and with only Hoechst 33342 (magenta). The conformations of R14 and D142 at the end of each simulation are shown as a visual aid. d) In simulations without POPG present (pink and magenta) the distance between R14 and D142 increases above that observed in the crystal structure. When POPG is present (teal and cyan) the distance between R14 and D142 is consistent with that observed in the crystal structure. Two independent simulations for each simulation condition are shown.

Figure 4.. Native mass spectra of LmrP and the N116Y mutant reconstituted in nanodiscs made of DOPE (80%) and DOPG (20%).

a) At low activation energy (160V) we observe multiple lipids bound to LmrP, including a peak corresponding to a single DOPG bound (in red). At high energy (200V) this peak is still present. In the case of the N116Y mutant, while we observe a bound DOPG at low energy, it disappears at high activation energy. b) Residue 116 is positioned inside the protein, and mutation to tyrosine (as depicted by the placement of a common tyrosine rotamer, yellow spheres) is expected to perturb the binding of the interior lipid (which is shown by the corresponding 2F_o-F_c map at 1 σ).

Figure 5.. The S52Y, T56Y and N116Y mutations alter substrate specificity without directly interacting with Hoechst 33342.

a) Transport of Hoechst 33342 is significantly reduced in the T56Y (purple) and N116Y (blue) mutants compared to wild-type LmrP (black). b) Cell survival assay demonstrating S52Y (green), T56Y (purple) and N116Y (blue) LmrP mutants cannot grow in as high a concentration of tetracycline as wild-type LmrP (black). c) Cell survival assay demonstrating S52Y (green), T56Y (purple) and N116Y (blue) LmrP mutants cannot grow in as high a concentration of erythromycin as wild-type LmrP (black). d) Cell survival assay showing that S52Y (green), T56Y (purple) and N116Y (blue) LmrP mutants are able to grow in equivalent concentrations of clindamycin to wild-type LmrP (black). The non-functional LmrP mutant D68N (red) is included as a negative control in panels. Chemical structures of Hoechst 33342, tetracycline, erythromycin and clindamycin are presented alongside the accompanying data. Error bars in panels b, c and d show standard-error mean (n=6).