Lipid composition regulates the orientation of transmembrane helices in HorA, an ABC multidrug transporter

Abstract

ATP-binding cassette (ABC) transporters constitute a large class of molecular pumps whose central role in chemotherapy resistance has highlighted their clinical relevance. We investigated whether the lipid composition of the membrane affects the function and structure of HorA, a bacterial ABC multidrug transporter. When the transporter was reconstituted in a bilayer where phosphatidylethanolamine (PE), the main lipid of the bacterial membrane, was replaced with phosphatidylcholine (PC), ATP hydrolysis and substrate transport became uncoupled. Although ATPase activity was maintained, HorA lost its ability to extrude the prototypical substrate Hoechst33342. Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed that, although the secondary structure of the protein was unaffected, the orientation of the transmembrane helices (TM) was modified by the change in lipid composition. The orientation of the backbone carbonyls indicated that the helices opened wider in PE versus PC-containing liposomes, with 10 degrees difference. This was supported by hydrogen/deuterium exchange studies showing increased protection of the backbone from the solvent in PC-containing liposomes. Electron Paramagnetic Resonance was used to further probe the structural change. In the PC-containing liposomes we observed increased mobility of the spin label in TM4, along with increased exposure to molecular oxygen, used as a hydrophobic quencher. This indicates that the lipid change induced modification of the orientation of TM4, exposing Cys-180 to the lipid phase. The lipid composition of the bilayer thus modulates the structure of HorA, and in turn its ability to extrude its substrates.

FIGURE 1.

Effect of DOPE on HorA activity. A, ATPase activity. HorA proteoliposomes containing either DOPE or DOPC were incubated with 2 mm ATP. The specific ATPase activity was calculated by measuring the release of inorganic phosphate (see “Experimental Procedures”). The data are means of four independent experiments, with error bars representing S.D. B, Hoechst 33342 transport. PE and PC proteoliposomes (about 15 μg of protein) were diluted to 950 μl. After 3 min, Hoechst 33342 was added to the suspension. Once the Hoechst fluorescence had reached a steady level, ATP was added (arrow) to a final concentration of 2 mm. The fluorescence intensity before addition of ATP was normalized to 100%.

FIGURE 2.

Secondary structure comparison. Eight independent ATR-FTIR spectra of reconstituted HorA were recorded for both PE and PC liposomes. The figure shows the Amide I region of the average ATR-FTIR spectra. No significant differences appear between the two mean spectra.

FIGURE 3.

Dichroism spectra. A, ATR-FTIR spectra of reconstituted HorA were recorded with incident light polarized perpendicularly (i) or parallel (ii) to the germanium plate. The dichroism spectrum (iii) was obtained by subtraction of the parallel-polarized spectrum from the perpendicularly polarized spectrum as described under “Experimental Procedures.” The Amide I region is highlighted. The vertical scale is given in reference to spectrum (i) and, for clarity, spectrum (ii) and (iii) were expanded by coefficients of 1.93 and 5.23, respectively. B, dichroism spectra of HorA reconstituted in both PE liposomes (a) and PC liposomes (b) show a positive deviation in the Amide I region, with a maximum in the specific region of the α-helices (marked).

FIGURE 4.

Kinetic of H/D exchange. A, spectra of PE and PC proteoliposomes were recorded as a function of time of exposure to a D₂O-saturated N₂ flux. B, deuteration leads to a decrease of the amide II band intensity. C, proportion of unexchanged amide bonds in HorA was monitored during deuteration in both PE (●) and PC proteoliposomes (○). It was calculated from the area of the amide II band during deuteration. The data are means of three independent experiments, with error bars representing S.D.

FIGURE 5.

EPR spectra of HorA labeled on Cys-180. A, sequence alignment of TM4 from HorA and Sav1866, showing that Cys-180 corresponds to Thr-177 in Sav1866. B, location of Thr-177 in the Sav1866 homodimer structure. C, EPR spectra of MTSSL-labeled HorA reconstituted in PE and PC proteoliposomes. Replacement of DOPC with DOPE induces the appearance of a mobile component (marked) in the EPR spectral line shape. D, accessibility of the MTSSL probe was assessed by quenching with molecular oxygen (filled bars) or NiEDDA (empty bars). Data are means of three independent experiments, with error bars representing S.D.