Abstract
Lipids are critical components of membranes that could affect the properties of membrane proteins, yet the precise compositions of lipids surrounding membrane-embedded protein complexes is often difficult to discern. Here we report that, for the heterodimeric ABC transporter TmrAB, the extent of delipidation can be controlled by timed exposure to detergent. We subsequently characterize the cohort of endogenous lipids that are extracted in contact with the membrane protein complex, and show that with prolonged delipidation the number of neutral lipids is reduced in favour of their negatively charged counterparts. We show that lipid A is retained by the transporter and that the extent of its binding decreases during the catalytic cycle, implying that lipid A release is linked to adenosine tri-phosphate hydrolysis. Together, these results enable us to propose that a subset of annular lipids is invariant in composition, with negatively charged lipids binding tightly to TmrAB, and imply a role for this exporter in glycolipid translocation.
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References
Bao, H., Dalal, K., Wang, V., Rouiller, I. & Duong, F. The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity. Biochim. Biophys. Acta 1828, 1723–1730 (2013).
Dowhan, W. & Bogdanov, M. Lipid–protein interactions as determinants of membrane protein structure and function. Biochem. Soc. Trans. 39, 767–774 (2011).
Betaneli, V., Petrov, E. P. & Schwille, P. The role of lipids in VDAC oligomerization. Biophys. J. 102, 523–531 (2012).
Obara, K. et al. Structural role of countertransport revealed in Ca2+ pump crystal structure in the absence of Ca2+. Proc. Natl Acad. Sci. USA 102, 14489–14496 (2005).
Laganowsky, A. et al. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175 (2014).
Lee, A. G. Biological membranes: the importance of molecular detail. Trends Biochem. Sci. 36, 493–500 (2011).
Lee, A. G. Lipid–protein interactions in biological membranes: a structural perspective. Biochim. Biophys. Acta 1612, 1–40 (2003).
Schweizer, H. P. Understanding efflux in Gram-negative bacteria: opportunities for drug discovery. Exp. Opin. Drug Discov. 7, 633–642 (2012).
Huang, Y. & Sadee, W. Membrane transporters and channels in chemoresistance and -sensitivity of tumor cells. Cancer Lett. 239, 168–182 (2006).
Gottesman, M. M., Fojo, T. & Bates, S. E. Multidrug resistance in cancer: role of ATP-dependent transporters. Nature Rev. Cancer 2, 48–58 (2002).
Borst, P., Zelcer, N. & van Helvoort, A. ABC transporters in lipid transport. Biochim. Biophys. Acta 1486, 128–144 (2000).
Reuter, G. et al. The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities. J. Biol. Chem. 278, 35193–35198 (2003).
Hendrich, A. B. & Michalak, K. Lipids as a target for drugs modulating multidrug resistance of cancer cells. Curr. Drug Targets 4, 23–30 (2003).
King, G. & Sharom, F. J. Proteins that bind and move lipids: MsbA and NPC1. Crit. Rev. Biochem. Mol. Biol. 47, 75–95 (2012).
Tarling, E. J., de Aguiar Vallim, T. Q. & Edwards, P. A. Role of ABC transporters in lipid transport and human disease. Trends Endocrinol. Metab. 24, 342–350 (2013).
Shintre, C. A. et al. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proc. Natl Acad. Sci. USA 110, 9710–9715 (2013).
Doerrler, W. T. & Raetz, C. R. ATPase activity of the MsbA lipid flippase of Escherichia coli. J. Biol. Chem. 277, 36697–36705 (2002).
Marek, M. et al. The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity. J. Biol. Chem. 286, 21835–21843 (2011).
Eggensperger, S., Fisette, O., Parcej, D., Schäfer, L. V. & Tampé, R. An annular lipid belt is essential for allosteric coupling and viral inhibition of the antigen translocation complex TAP. J. Biol. Chem. http://dx.doi.org/10.1074/jbc.M114.592832 (2014).
Barrera, N. P., Di Bartolo, N., Booth, P. J. & Robinson, C. V. Micelles protect membrane complexes from solution to vacuum. Science 321, 243–246 (2008).
Zhou, M. et al. Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334, 380–385 (2011).
Barrera, N. P. et al. Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nature Methods 6, 585–587 (2009).
Schmidt, C. et al. Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation. Nature Commun. 4, 1985 (2013).
Marcoux, J. et al. Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc. Natl Acad. Sci. USA 110, 9704–9709 (2013).
Zutz, A. et al. Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus. J. Biol. Chem. 286, 7104–7115 (2011).
Laganowsky, A., Reading, E., Hopper, J. T. & Robinson, C. V. Mass spectrometry of intact membrane protein complexes. Nature Protoc. 8, 639–651 (2013).
Kaltashov, I. A. & Mohimen, A. Estimates of protein surface areas in solution by electrospray ionization mass spectrometry. Anal. Chem. 77, 5370–5379 (2005).
Lomize, M. A., Lomize, A. L., Pogozheva, I. D. & Mosberg, H. I. OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006).
Morgner, N. & Robinson, C. V. Massign: an assignment strategy for maximizing information from the mass spectra of heterogeneous protein assemblies. Anal. Chem. 84, 2939–2948 (2012).
Lukasiewicz, J., Jachymek, W., Niedziela, T., Kenne, L. & Lugowski, C. Structural analysis of the lipid A isolated from Hafnia alvei 32 and PCM 1192 lipopolysaccharides. J. Lipid Res. 51, 564–574 (2010).
Raetz, C. R. et al. Kdo2-lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4. J. Lipid Res. 47, 1097–1111 (2006).
Lewis, B. A. & Engelman, D. M. Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles. J. Mol. Biol. 166, 211–217 (1983).
Palsdottir, H. & Hunte, C. Lipids in membrane protein structures. Biochim. Biophys. Acta. 1666, 2–18 (2004).
Lee, A. G. How lipids and proteins interact in a membrane: a molecular approach. Mol. Biosyst. 1, 203–212 (2005).
Yeagle, P. L. Non-covalent binding of membrane lipids to membrane proteins. Biochim. Biophys. Acta 1838, 1548–1559 (2014).
Lemieux, M. J., Reithmeier, R. A. & Wang, D. N. Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain. J. Struct. Biol. 137, 322–332 (2002).
Clay, A. T. & Sharom, F. J. Lipid bilayer properties control membrane partitioning, binding, and transport of p-glycoprotein substrates. Biochemistry 52, 343–354 (2013).
Dong, J., Yang, G. & McHaourab, H. S. Structural basis of energy transduction in the transport cycle of MsbA. Science 308, 1023–1028 (2005).
Woebking, B. et al. Functional role of transmembrane helix 6 in drug binding and transport by the ABC transporter MsbA. Biochemistry 47, 10904–10914 (2008).
Woebking, B. et al. Drug–lipid A interactions on the Escherichia coli ABC transporter MsbA. J. Bacteriol. 187, 6363–6369 (2005).
Hohl, M., Briand, C., Grutter, M. G. & Seeger, M. A. Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation. Nature Struct. Mol. Biol. 19, 395–402 (2012).
Lubelski, J., van Merkerk, R., Konings, W. N. & Driessen, A. J. Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric. Biochemistry 45, 648–656 (2006).
Yang, R., Cui, L., Hou, Y. X., Riordan, J. R. & Chang, X. B. ATP binding to the first nucleotide binding domain of multidrug resistance-associated protein plays a regulatory role at low nucleotide concentration, whereas ATP hydrolysis at the second plays a dominant role in ATP-dependent leukotriene C4 transport. J. Biol. Chem. 278, 30764–30771 (2003).
Schölz, C. et al. Specific lipids modulate the transporter associated with antigen processing (TAP). J. Biol. Chem. 286, 13346–13356 (2011).
Ward, A. B., Guvench, O. & Hills, R. D. Jr. Coarse grain lipid–protein molecular interactions and diffusion with MsbA flippase. Proteins 80, 2178–2190 (2012).
Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C. & Raetz, C. R. Function of Escherichia coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis. J. Biol. Chem. 273, 12466–12475 (1998).
Doerrler, W. T., Reedy, M. C. & Raetz, C. R. An Escherichia coli mutant defective in lipid export. J. Biol. Chem. 276, 11461–11464 (2001).
Rees, D. C., Johnson, E. & Lewinson, O. ABC transporters the power to change. Nature Rev. Mol. Cell Biol. 10, 218–227 (2009).
Ryan, C. M. et al. Post-translational modifications of integral membrane proteins resolved by top-down Fourier transform mass spectrometry with collisionally activated dissociation. Mol. Cell. Proteom. 9, 791–803 (2010).
Rosati, S., Yang, Y., Barendregt, A. & Heck, A. J. Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry. Nature Protoc. 9, 967–976 (2014).
Tseng, W. C., Lin, J. W., Wei, T. Y. & Fang, T. Y. A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal. Biochem. 375, 376–378 (2008).
Sobott, F., Hernandez, H., McCammon, M. G., Tito, M. A. & Robinson, C. V. A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal. Chem. 74, 1402–1407 (2002).
Hernandez, H. & Robinson, C. V. Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nature Protoc. 2, 715–726 (2007).
Oursel, D. et al. Lipid composition of membranes of Escherichia coli by liquid chromatography/tandem mass spectrometry using negative electrospray ionization. Rapid Commun. Mass Spectrom. 21, 1721–1728 (2007).
Jones, J. W., Shaffer, S. A., Ernst, R. K., Goodlett, D. R. & Turecek, F. Determination of pyrophosphorylated forms of lipid A in Gram-negative bacteria using a multivaried mass spectrometric approach. Proc. Natl Acad. Sci. USA 105, 12742–12747 (2008).
Acknowledgements
The authors thank C. Schmidt for help with the LC-MS experiments and all the members of C.V.R.'s group for stimulating discussions. The authors also acknowledge funding from European Research Council Integral Membrane Proteins Resolution of Stoichiometry and Structure (ERC IMPRESS), the Royal Society and the Germany Research Foundation (SFB 807 and TA157/7 to R.T.) as well as the European Drug Initiative on Channels and Transporters (EDICT to R.T.) funded by the European Commission Seventh Framework. M.T.D. is supported by the Swiss National Science Foundation.
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C.V.R., C.B and R.T. conceived and designed the research. C.B. devised the delipidation protocol, conducted the MS experiments and analysed the data. A.N. expressed and purified TmrAB and performed biochemical analyses. N.M. ran simulations and the fitting of mass spectra. M.T.D. designed and ran MD simulations. C.V.R., C.B. and R.T. wrote the paper, with contributions from all co-authors.
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Bechara, C., Nöll, A., Morgner, N. et al. A subset of annular lipids is linked to the flippase activity of an ABC transporter. Nature Chem 7, 255–262 (2015). https://doi.org/10.1038/nchem.2172
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DOI: https://doi.org/10.1038/nchem.2172
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