Letter | Published:

Membrane proteins bind lipids selectively to modulate their structure and function

Nature volume 510, pages 172175 (05 June 2014) | Download Citation

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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Accessions

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

Author notes

    • Arthur Laganowsky
    •  & Eamonn Reading

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 5QY, UK

    • Arthur Laganowsky
    • , Eamonn Reading
    • , Timothy M. Allison
    • , Matteo T. Degiacomi
    • , Andrew J. Baldwin
    •  & Carol V. Robinson
  2. Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA

    • Martin B. Ulmschneider

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Arthur Laganowsky or Carol V. Robinson.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains the Supplementary Discussion and a Supplementary Reference.

Videos

  1. 1.

    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.

  2. 2.

    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.

  3. 3.

    Gas-phase unfolding of ApqZ bound to PC

    Animation created and shown as described in Supplementary video 2.

  4. 4.

    Gas-phase unfolding of AmtB bound to PG

    Animation created and shown as described in Supplementary Video 2.

  5. 5.

    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.

  6. 6.

    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).

  7. 7.

    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.

  8. 8.

    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.

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DOI

https://doi.org/10.1038/nature13419

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