Abstract
Direct interactions with lipids have emerged as key determinants of the folding, structure and function of membrane proteins, but an understanding of how lipids modulate protein dynamics is still lacking. Here, we systematically explored the effects of lipids on the conformational dynamics of the proton-powered multidrug transporter LmrP from Lactococcus lactis, using the pattern of distances between spin-label pairs previously shown to report on alternating access of the protein. We uncovered, at the molecular level, how the lipid headgroups shape the conformational-energy landscape of the transporter. The model emerging from our data suggests a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein. Together, our data lay the foundation for a comprehensive model of secondary multidrug transport in lipid bilayers.
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Acknowledgements
We thank D. Claxton, R. Dastvan and D. Hilger for critically reading the manuscript, V. Debruycker (ULB) for providing the solubilized protein used for the MS measurements and J.M. Ruysschaert for helpful discussions. This work was supported by the Fonds de la Recherche Scientifique F.R.S.–F.N.R.S. (grant F.4523.12 to C.G.) and the NIH (grant R01GM077659 to H.S.M.) C.M. is supported as a Research Fellow of the F.R.I.A. C.G. is supported as a Research Associate of the F.R.S.–F.N.R.S. and as a Welbio Investigator.
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C.M., H.S.M. and C.G. performed experimental design. M.M., S.M., A.R. and C.M. performed mutagenesis, expression, activity, purification, reconstitution and labeling experiments. R.A.S. performed EPR measurements. R.A.S., C.M. and H.S.M. performed DEER data analysis. C.G. and C.M. performed molecular modeling. C.M. and A.R. performed transport assays. A.K. and F.S. performed native MS measurements and data analysis. R.D. performed LC-MS/MS measurements and analysis. C.G. and H.S.M. oversaw all aspects of the experiments and manuscript preparation. All authors participated in interpreting the data and writing the paper.
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Integrated supplementary information
Supplementary Figure 1 Limited ligand-dependent conformational changes within the N or C lobe of LmrP.
DEER distance distributions for spin-labeled cysteine pairs located on the extracellular (left panels) and cytoplasmic (right panels) at ends of TM helices, within a same lobe, at pH8 (black), pH6 (red), pH8 + Hoechst 33342 (blue) and pH8 + EtBr (orange). Residue numbers and positions are depicted on an LmrP model viewed from the extracellular (top left) or cytoplasmic (top right) side. The N-lobe is blue and the C-lobe is grey.
Supplementary Figure 2 Transport activity assay of double-cysteine mutants.
LmrP-dependent Hoechst 33342 extrusion is monitored in inside-out membrane vesicles by a fluorescence-based assay (see Methods). Values were normalized for fluorescence intensity and total protein amount. Addition of ATP activates the endogenous F0/F1 – ATPase leading to formation of a transmembrane proton gradient, thus triggering LmrP activity. All the double cysteine mutants retained significant activity, compared to the D68C mutant used as a negative control.
Supplementary Figure 3 EtBr binding induces minor conformational changes.
DEER distance distributions for spin-labeled cysteine pairs located on the extracellular (top panels) and cytoplasmic (bottom panels) ends of TM helices. No significant changes are observed at pH8 in the absence (black) and in presence of 1mM EtBr (orange), except for the 256R1-310R1 pair, reporting substrate binding. For this pair, substrate binding leads to narrowing of the distance distribution. Targeted helices are highlighted in orange with TM numbers indicated atop, on an LmrP model viewed from the extracellular (top) or cytoplasmic (bottom) side. The N-lobe is blue and the C-lobe is grey.
Supplementary Figure 4 Uncoupling of the charge-relay network in detergent.
(a) Crystal structure of the LmrP homolog YajR in the outward-open conformation. The charge-relay network of conserved residues is highlighted. TM2 and TM11 are displayed in orange. (b). the single mutations D68N, R72K and D128N were combined with extracellular (160R1-310R1) and (c) cytoplasmic (137R1-349R1) reporters. DEER distance distributions at pH5 (red) pH8 (black) and pH8 + Hoechst 33342 (blue) in the absence (dashed line) and presence (solid line) of each mutation.
Supplementary Figure 5 Phospholipid headgroup modulates the pH-dependent conformational equilibrium.
DEER distance distributions of the 160R1-310R1 (upper half) and the 137R1-349R1 (lower half) pairs, used as extracellular and cytoplasmic distance reporters, respectively, reconstituted in nanodiscs composed of DOPE-DOPG-CL (grey), DOPE(Me)2-DOPG-CL (blue) and DOPC-DOPG-CL (orange), at four pHs values ranging from 5 to 8.
Supplementary Figure 6 Various endogenous cardiolipin species bind to LmrP.
(a) Mass spectrum of detergent-solubilized LmrP sample (native MS). Red rectangle: Cardiolipin molecules ejected from LmrP upon increased voltage. Blue rectangle: LmrP with (red arrows) and without (blue labels) cardiolipin(s) bound. (b) Lipids quantification by liquid chromatography and tandem mass spectrometry (n=3, Error bars: s.e.m). Red: lipids extracted from L.lactis membranes. Blue: lipids co-purified with LmrP.
Supplementary Figure 7 The conformational equilibrium of LmrP is not affected by changes in the ionic strength.
DEER distance distributions of the 160R1-310R1 pair in E.coli polar lipids nanodiscs (left) and detergent micelles (right) at NaCl concentrations ranging from a third to the double of the usual concentration.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 (PDF 1271 kb)
Supplementary Data Set 1
EmrD based homology model (TXT 245 kb)
Supplementary Data Set 2
LacY based homology model (TXT 245 kb)
Supplementary Data Set 3
FucP based homology model (TXT 167 kb)
Supplementary Data Set 4
YajR based homology model (TXT 247 kb)
Supplementary Data Set 5
Continuous-wave EPR spectra (TIFF 237 kb)
Supplementary Data Set 6
DEER data analysis (TIFF 423 kb)
Supplementary Data Set 7
DEER data analysis continued (TIFF 467 kb)
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Martens, C., Stein, R., Masureel, M. et al. Lipids modulate the conformational dynamics of a secondary multidrug transporter. Nat Struct Mol Biol 23, 744–751 (2016). https://doi.org/10.1038/nsmb.3262
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DOI: https://doi.org/10.1038/nsmb.3262
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