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Membrane proteins bind lipids selectively to modulate their structure and function

Abstract

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments1,2,3,4,5,6,7 and that lipids can bind to specific sites, for example, in potassium channels8. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation9. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

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Figure 1: The mechanosensitive channel of large conductance (MscL) resists unfolding in the presence of lipids.
Figure 2: AqpZ is indiscriminately stabilized by lipids with the exception of cardiolipin, a lipid that stabilizes the channel significantly and modulates its function.
Figure 3: Lipid binding to AmtB results in a range of stabilizing effects.
Figure 4: Crystal structure of AmtB bound to PG.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the crystal structure have been deposited with the Protein Data Bank (PDB) under accession code 4NH2.

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Acknowledgements

We thank J. Hobman and D. Lee for providing gene-doctoring plasmids, Z. Guan and C. Li for providing E. coli strains and plasmids containing cardiolipin genes. We also thank D. Staunton and N. Housden for training on the stopped-flow apparatus; and T. Mize, J. Benesch, M. McDonough, T. Walton and D. Rees for discussions. We also gratefully acknowledge E. Lowe and S. Lea for organizing synchrotron proposals and Diamond Light Source beam line I04 and staff, the Medical Research Council (MRC), BBSRC and ERC advanced grant (IMPRESS) for funding. A.L. is a Nicholas Kurti Junior Research Fellow of Brasenose College, A.J.B. is a BBSRC David Phillip’s Fellow and C.V.R. is a Royal Society Professor.

Author information

Authors and Affiliations

Authors

Contributions

A.L., E.R., and C.V.R. designed the research. A.L. and E.R. performed the experiments. T.M.A. assisted A.L. and E.R. in protein expression and purification. M.B.U. carried out molecular dynamics. M.T.D., A.J.B. and A.L. designed and performed post-molecular dynamics analyses. A.L., E.R., T.M.A. and A.J.B. developed IM-MS analysis software. A.L. and E.R. analysed the data. A.L., E.R. and C.V.R. wrote the paper with input from the other authors.

Corresponding authors

Correspondence to Arthur Laganowsky or Carol V. Robinson.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Maintaining intact native membrane protein complexes in the mass spectrometer.

a, b, Gas-phase unfolding plots (left, 5 V steps) and mass spectra (right) of detergent stripped AqpZ and AmtB ions (charge inset) from different non-ionic detergent solutions; C8E4 (pink), NG (green), OGNG (purple) and DDM (orange). Membrane protein complexes from NG, OGNG and DDM possessed CCS values substantially larger than those calculated from crystal structures. c, Complete removal of C8E4 at low-collision voltages reveals CCS values consistent with those calculated from their respective crystal structures. d, Reported are measured masses, standard deviations, and empirical t0 values used for direct CCS calculation of membrane proteins studied.

Extended Data Figure 2 Phospholipid abbreviations and optimization of phospholipid binding experiments.

a, Phospholipid abbreviations and headgroup structures. b, Protein to phospholipid to detergent ratios (P:L:D) for each membrane protein, number of resolvable phospholipids bound with their masses. Conditions were optimized empirically to maintain nanospray, sufficient mass spectral quality and phospholipid binding. Masses for one lipid bound to the protein complex were measured using MassLynx V4.1 software (Waters).

Extended Data Figure 3 Phospholipid binding to MscL reveals an insignificant impact on protein gas-phase stabilization independent of lipid alkyl chain length.

a, Representative mass and ion mobility spectra of MscL bound to various phospholipids species (inset top right). b, Representative mass and ion mobility spectra of MscL bound to phospholipids of varying alkyl chain length. c, Cumulative stabilization of MscL is seen for one (blue) to two (red) bound lipid molecules. Reported are the means ± s.e.m. from repeated measurements (n = 3).

Extended Data Figure 4 Summary of statistics for molecular dynamics (MD) simulations followed by CCS filtering.

a, Representative filtering procedure of AqpZ in a PC bilayer from the MD (top). For each frame, lipids are extracted within 6 Å of the protein (bottom left) then filtered by the experimental CCS of AqpZ bound to a single PC molecule (bottom right); see Methods. b, Summary of statistics. c, Ratios of phospholipid bound to apo CCS values for membrane protein complexes. Reported are the mean and standard deviation (n = 3). d, MD filtered by CCS resulted in similar patches of PC molecules across different time points within the simulation indicating the systems were equilibrated.

Extended Data Figure 5 MD simulations filtered by CCS reveals probable one (1×) and two (2×) phospholipid binding sites.

a, For each candidate structure, protein and 1× or 2× PC molecule(s), the ratio of their calculated CCS values was determined (CCS ratio). This procedure generated a large number of candidate structures (grey bars) that were filtered using our CCS measurements (cyan line). The structures in grey that intercept this curve are essentially the ones selected as our most probable ensemble. b, The intersection between the simulated lipid complexes and the experimental data are then projected onto the surface of the protein to identify the most probable binding sites. Probable lipid locations for MscL resembled an annular belt, with no specific patches of lipids probably stemming from the relatively cylindrical geometry of this complex. By contrast, for AqpZ and AmtB the most probable location of the lipid molecules were localized to the interfacial regions between protein subunits, as well as other probable locations on individual monomers. c, X-ray derived PG (blue spheres with white tails) located at the subunit interfaces agrees with the predicted PC binding site.

Extended Data Figure 6 Modelling and quantification of gas-phase unfolding pathways.

a, Representative ion mobility mass spectra are collected over a range of collision voltages. b, Ion arrival time is converted to CCS before generating unfolding plots. c, Model fitting process from 2D to 3D data (see Methods). d, Both a Synapt2 modified with a linear drift cell (DT-IMS) and the commercially available travelling wave SynaptG2 (TW-IMS) produce qualitatively similar unfolding data. e, A contour plot representing the variance of CCS of two gas-phase unfolding species as a function of ion mobility drift cell potential. f, Stacked plots of arrival time distributions for two gas-phase unfolding species as a function of drift cell potential. The lifetime of unfolding protein complexes in the drift tube ranges from 4 to 15 ms depending on the drift cell potential. No additional unfolding post activation occurs implying that the unfolding mechanism is not consistent with an irreversible unfolding model. Such a mechanism would predict time dependence on the population of unfolded species. By contrast the unfolding mechanism is well described by the reversible unfolding mechanism (see Methods).

Extended Data Figure 7 AqpZ, AmtB and AmtBN72A/N79A bound to various phospholipid species.

a, Representative mass and ion mobility spectra of AqpZ bound to phospholipids. b, Representative mass and ion mobility spectra of AmtB bound to phospholipids and AmtBN72A/N79A bound to PG.

Extended Data Figure 8 Summary of water permeability assays and analysis of lipid extracts.

a, HPTLC analysis of total polar lipid extract from wild-type E. coli (EPL), cardiolipin-deficient strain (BKT22), or BKT22 cells expressing ClsC and YmdB (BKT22-YC) to restore cardiolipin. Lipids were quantified by densiometry. b, Reported are the rate constants (kwat) and standard error of replicates (n = 5) for empty liposomes (−) and AqpZ proteoliposomes (+) reconstituted in differing E. coli lipid compositions.

Extended Data Figure 9 Structural analysis of AmtB bound to PG.

a, Crystal packing with six AmtB (multicoloured) and eight PG (orange) molecules located in the asymmetric unit cell and symmetry related molecules shown in grey and light orange, respectively. b, FoFc and 2FoFc electron density maps after refinement without lipid and near-lipid water molecule (if present) contoured at 2.0 and 1.0 sigma, respectively. c, Comparison of AmtB bound to PG (green chain, this work) aligned with the AmtB structure (maroon chain, PDB: 1U7G). d, Structure overlay of AmtB–GlnK complex bound to octylglucoside (purple chain, PDB: 2NS1) aligned with AmtB bound to PG reveals a distinct conformational change. The lipid–water interface (grey plane) was determined from coordinates of phosphate atoms from bound PG.

Extended Data Table 1 Summary of X-ray data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains the Supplementary Discussion and a Supplementary Reference. (PDF 110 kb)

Animation of an ion mobility mass spectrometer.

Purified membrane proteins (about 1-3 uL in high nM range) are subjected to nano electrospray ionization in positive mode that results in a distribution of positively charged protein-detergent complexes or ions. These ions enter the mass spectrometer and pass through a series of ion optics, such as the quadrupole. Next, ions enter the collision cell where they are accelerated into inert gas (typically argon) to emerge membrane proteins from the micelle as well as to unfold them in the gas-phase. After the collision cell, ions enter the ion mobility cell to perform a separation based on their shape (rotationally averaged collision cross section) through interaction with a carrier gas (helium or nitrogen). Shown is ion mobility separation of native aquaporin Z in cartoon representation and helium atoms as blue spheres. Post ion mobility, ions pass through ion optics to the time-of-flight mass analyser to obtain a high-resolution mass measurement. Instrument parts and the typical working ranges for membrane protein complexes are shown. (MOV 21015 kb)

Gas-phase unfolding of MscL bound to PI

Ion mobility mass spectra are shown in linear scale and colour-coded as shown in Fig. 1b. Animation was created by linear interpolation between ion mobility mass spectra collected every five collision volts (a feature of our software). Shown in the inset is the collision voltage. (MOV 11195 kb)

Gas-phase unfolding of ApqZ bound to PC

Animation created and shown as described in Supplementary video 2. (MOV 7986 kb)

Gas-phase unfolding of AmtB bound to PG

Animation created and shown as described in Supplementary Video 2. (MOV 10303 kb)

Morph of AmtB PG- to OG-bound structure

Structural morph was created using the morph command in Pymol71 with the structure reported here and pdb 1U7G. (MOV 2805 kb)

MD simulation of MscL in PC bilayer with lipids colour-coded by their agreement with CCS measurement for MscL bound to one PC molecule

Lipids follow the colour scheme for relative probability as shown in Extended Data Figure 5b. Animated are 0.2 ns snapshots over the entire simulation (Extended Data Figure 4). (MOV 27655 kb)

MD simulation of AqpZ in PC bilayer with lipids colour-coded by their agreement with CCS measurement for AqpZ bound to one PC molecule

Lipids are shown as described in Supplementary Video 6. Animation was created as described in Supplementary Video 6. (MOV 27634 kb)

MD simulation of AmtB in PC bilayer with lipids colour-coded by their agreement with CCS measurement for AmtB bound to one PC molecule

Lipids are shown as described in Supplementary Video 6. Animation was created as described in Supplementary Video 6. (MOV 27666 kb)

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Laganowsky, A., Reading, E., Allison, T. et al. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175 (2014). https://doi.org/10.1038/nature13419

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