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A subset of annular lipids is linked to the flippase activity of an ABC transporter

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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Figure 1: Progressive delipidation of TmrAB monitored by nano-electrospray mass spectrometry.
Figure 2: Phospholipids bound to TmrAB.
Figure 3: Molecular dynamics simulations of inward and outward models of TmrAB in a lipid bilayer.
Figure 4: Lipid A species bound to TmrAB.
Figure 5: Displacement of lipid A from proteomicelles upon ATP hydrolysis.

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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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Correspondence to Robert Tampé or Carol V. Robinson.

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The authors declare no competing financial interests.

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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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