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Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein

Abstract

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

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Figure 1: SSNMR spectra of ASR recorded on sparsely labeled samples.
Figure 2: Schematic representation of iterative long-range cross-peak assignment and convergence of structure calculation.
Figure 3: Overall topology and inter-monomer packing of ASR trimer.
Figure 4: Structural organization of ASR monomer.
Figure 5: A comparison of ASR structures in 3D crystals and in lipids.

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References

  1. Stroud, R.M. New tools in membrane protein determination. F1000 Biol. Rep. 3, 8 (2011).

    PubMed  PubMed Central  Article  Google Scholar 

  2. Bill, R.M. et al. Overcoming barriers to membrane protein structure determination. Nat. Biotechnol. 29, 335–340 (2011).

    CAS  PubMed  Article  Google Scholar 

  3. Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).

    CAS  PubMed  Article  Google Scholar 

  4. Cherezov, V. et al. High-resolution crystal structure of an engineered human beta2-adrenergic G protein–coupled receptor. Science 318, 1258–1265 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Rasmussen, S.G. et al. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450, 383–387 (2007).

    CAS  Article  PubMed  Google Scholar 

  6. Jaakola, V.P. et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211–1217 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Shimamura, T. et al. Structure of the human histamine H1 receptor complex with doxepin. Nature 475, 65–70 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Kim, H.J., Howell, S.C., Van Horn, W.D., Jeon, Y.H. & Sanders, C.R. Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins. Prog. Nucl. Magn. Reson. Spectrosc. 55, 335–360 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Hiller, S. & Wagner, G. The role of solution NMR in the structure determinations of VDAC-1 and other membrane proteins. Curr. Opin. Struct. Biol. 19, 396–401 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Nietlispach, D. & Gautier, A. Solution NMR studies of polytopic alpha-helical membrane proteins. Curr. Opin. Struct. Biol. 21, 497–508 (2011).

    CAS  PubMed  Article  Google Scholar 

  11. Zhou, Y. et al. NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation. Mol. Cell 31, 896–908 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Van Horn, W.D. et al. Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324, 1726–1729 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Gautier, A., Mott, H.R., Bostock, M.J., Kirkpatrick, J.P. & Nietlispach, D. Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy. Nat. Struct. Mol. Biol. 17, 768–774 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Reckel, S. et al. Solution NMR structure of proteorhodopsin. Angew. Chem. Int. Edn. Engl. 50, 11942–11946 (2011).

    CAS  Article  Google Scholar 

  15. Berardi, M.J., Shih, W.M., Harrison, S.C. & Chou, J.J. Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching. Nature 476, 109–113 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Lange, A. et al. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature 440, 959–962 (2006).

    CAS  PubMed  Article  Google Scholar 

  17. Ahuja, S. et al. Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation. Nat. Struct. Mol. Biol. 16, 168–175 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Sharma, M. et al. Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer. Science 330, 509–512 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Cady, S.D. et al. Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 463, 689–692 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Shahid, S.A. et al. Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals. Nat. Methods 9, 1212–1217 (2012).

    CAS  PubMed  Article  Google Scholar 

  21. Park, S.H. et al. Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491, 779–783 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Manolikas, T., Herrmann, T. & Meier, B.H. Protein structure determination from 13C spin-diffusion solid-state NMR spectroscopy. J. Am. Chem. Soc. 130, 3959–3966 (2008).

    CAS  PubMed  Article  Google Scholar 

  23. Loquet, A. et al. 3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints. J. Am. Chem. Soc. 130, 3579–3589 (2008).

    CAS  PubMed  Article  Google Scholar 

  24. Jung, K.H., Trivedi, V.D. & Spudich, J.L. Demonstration of a sensory rhodopsin in eubacteria. Mol. Microbiol. 47, 1513–1522 (2003).

    CAS  PubMed  Article  Google Scholar 

  25. Wang, S., Kim, S.Y., Jung, K.H., Ladizhansky, V. & Brown, L.S. A eukaryotic-like interaction of soluble cyanobacterial sensory rhodopsin transducer with DNA. J. Mol. Biol. 411, 449–462 (2011).

    CAS  PubMed  Article  Google Scholar 

  26. Vogeley, L. et al. Anabaena sensory rhodopsin: a photochromic color sensor at 2.0 Å. Science 306, 1390–1393 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Shi, L., Kawamura, I., Jung, K.H., Brown, L.S. & Ladizhansky, V. Conformation of a seven-helical transmembrane photosensor in the lipid environment. Angew. Chem. Int. Edn. Engl. 50, 1302–1305 (2011).

    CAS  Article  Google Scholar 

  28. Wang, S. et al. Paramagnetic relaxation enhancement reveals oligomerization interface of a membrane protein. J. Am. Chem. Soc. 134, 16995–16998 (2012).

    CAS  PubMed  Article  Google Scholar 

  29. Wang, S., Shi, L., Kawamura, I., Brown, L.S. & Ladizhansky, V. Site-specific solid-state NMR detection of hydrogen-deuterium exchange reveals conformational changes in a 7-helical transmembrane protein. Biophys. J. 101, L23–L25 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Peng, X., Libich, D., Janik, R., Harauz, G. & Ladizhansky, V. Dipolar chemical shift correlation spectroscopy for homonuclear carbon distance measurements in proteins in the solid state: application to structure determination and refinement. J. Am. Chem. Soc. 130, 359–369 (2008).

    CAS  PubMed  Article  Google Scholar 

  31. Lange, A., Luca, S. & Baldus, M. Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids. J. Am. Chem. Soc. 124, 9704–9705 (2002).

    CAS  PubMed  Article  Google Scholar 

  32. Castellani, F. et al. Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420, 98–102 (2002).

    CAS  PubMed  Article  Google Scholar 

  33. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    CAS  PubMed  Article  Google Scholar 

  34. Bardiaux, B., Malliavin, T. & Nilges, M. ARIA for solution and solid-state NMR. Methods Mol. Biol. 831, 453–483 (2012).

    CAS  PubMed  Article  Google Scholar 

  35. Nadaud, P.S., Helmus, J.J., Hofer, N. & Jaroniec, C.P. Long-range structural restraints in spin-labeled proteins probed by solid-state nuclear magnetic resonance spectroscopy. J. Am. Chem. Soc. 129, 7502–7503 (2007).

    CAS  Article  PubMed  Google Scholar 

  36. Essen, L., Siegert, R., Lehmann, W.D. & Oesterhelt, D. Lipid patches in membrane protein oligomers: crystal structure of the bacteriorhodopsin-lipid complex. Proc. Natl. Acad. Sci. USA 95, 11673–11678 (1998).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. Dencher, N.A. & Heyn, M.P. Formation and properties of bacteriorhodopsin monomers in the non-ionic detergents octyl-beta-D-glucoside and Triton X-100. FEBS Lett. 96, 322–326 (1978).

    CAS  PubMed  Article  Google Scholar 

  38. Shi, L. et al. Three-dimensional solid-state NMR study of a seven-helical integral membrane proton pump—structural insights. J. Mol. Biol. 386, 1078–1093 (2009).

    CAS  PubMed  Article  Google Scholar 

  39. Emami, S., Fan, Y., Munro, R., Ladizhansky, V. & Brown, L.S. Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra. J. Biomol. NMR 55, 147–155 (2013).

    CAS  PubMed  Article  Google Scholar 

  40. Klammt, C. et al. Facile backbone structure determination of human membrane proteins by NMR spectroscopy. Nat. Methods 9, 834–839 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. daCosta, C.J. & Baenziger, J.E. A rapid method for assessing lipid:protein and detergent:protein ratios in membrane-protein crystallization. Acta Crystallogr. D Biol. Crystallogr. 59, 77–83 (2003).

    PubMed  Article  CAS  Google Scholar 

  42. Morcombe, C.R. & Zilm, K.W. Chemical shift referencing in MAS solid state NMR. J. Magn. Reson. 162, 479–486 (2003).

    CAS  Article  PubMed  Google Scholar 

  43. Janik, R., Peng, X. & Ladizhansky, V. (13)C-(13)C distance measurements in U-(13)C, (15)N-labeled peptides using rotational resonance width experiment with a homogeneously broadened matching condition. J. Magn. Reson. 188, 129–140 (2007).

    CAS  PubMed  Article  Google Scholar 

  44. Delaglio, F. et al. NMRPipe—a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  PubMed  Article  Google Scholar 

  45. Keller, R. The Computer Aided Resonance Assignment Tutorial. 1st edn. (CANTINA Verlag Goldau, 2004).

  46. Wang, S. et al. Solid-state NMR (13)C and (15)N resonance assignments of a seven-transmembrane helical protein Anabaena Sensory Rhodopsin. Biomol. NMR Assign. doi:10.1007/s12104-012-9421-y (16 September 2012).

  47. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Tajkhorshid, E., Paizs, B. & Suhai, S. Conformational effects on the proton affinity of the Schiff base in bacteriorhodopsin: a density functional study. J. Phys. Chem. B 101, 8021–8028 (1997).

    CAS  Article  Google Scholar 

  49. Tajkhorshid, E., Baudry, J., Schulten, K. & Suhai, S. Molecular dynamics study of the nature and origin of retinal's twisted structure in bacteriorhodopsin. Biophys. J. 78, 683–693 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Tajkhorshid, E. & Suhai, S. Influence of the methyl groups on the structure, charge distribution, and proton affinity of the retinal Schiff base. J. Phys. Chem. B 103, 5581–5590 (1999).

    CAS  Article  Google Scholar 

  51. Baudry, J., Crouzy, S., Roux, B. & Smith, J.C. Quantum chemical and free energy simulation analysis of retinal conformational energetics. J. Chem. Inf. Comput. Sci. 37, 1018–1024 (1997).

    CAS  Article  Google Scholar 

  52. Farrar, M.R. et al. Solid-state NMR-study of [Epsilon-C-13]Lys-Bacteriorhodospin—Schiff-base photoisomerization. Biophys. J. 65, 310–315 (1993).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Smith, S.O. et al. Structure and protein environment of the retinal chromophore in light-adapted and dark-adapted bacteriorhodopsin studied by solid-state NMR. Biochemistry 28, 8897–8904 (1989).

    CAS  PubMed  Article  Google Scholar 

  54. Sammalkorpi, M. & Lazaridis, T. Modeling a spin-labeled fusion peptide in a membrane: implications for the interpretation of EPR experiments. Biophys. J. 92, 10–22 (2007).

    CAS  PubMed  Article  Google Scholar 

  55. Higman, V.A. et al. Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins. J. Biomol. NMR 44, 245–260 (2009).

    CAS  PubMed  Article  Google Scholar 

  56. Zech, S.G., Wand, A.J. & McDermott, A.E. Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin. J. Am. Chem. Soc. 127, 8618–8626 (2005).

    CAS  PubMed  Article  Google Scholar 

  57. Dominguez, C., Boelens, R. & Bonvin, A.M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 1731–1737 (2003).

    CAS  PubMed  Article  Google Scholar 

  58. de Vries, S.J. et al. HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets. Proteins 69, 726–733 (2007).

    CAS  PubMed  Article  Google Scholar 

  59. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK—a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993).

    CAS  Article  Google Scholar 

  60. Vriend, G. WHAT IF: a molecular modeling and drug design program. J. Mol. Graphics 8, 52–56 (1990).

    CAS  Article  Google Scholar 

  61. Rodriguez, R., Chinea, G., Lopez, N., Pons, T. & Vriend, G. Homology modeling, model and software evaluation: three related resources. Bioinformatics 14, 523–528 (1998).

    CAS  PubMed  Article  Google Scholar 

  62. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

This research was funded by Natural Science and Engineering Research Council of Canada, National Research Foundation of Korea (Global Research Network Program), Canada Foundation for Innovation and Ontario Ministry of Economic Development and Innovation. S.W. is a recipient of the Canadian Institutes for Health Research Postdoctoral Fellowship. V.L. is supported by Canada Research Chair in Biophysics (Tier II). We thank B. Bardiaux (Leibniz-Institut für Molekulare Pharmakologie, Berlin) for providing the ARIA 2.3 program before publication, and C.P. Jaroniec and J. Lanyi for carefully reading the manuscript.

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Contributions

S.W., L.S., I.K. and R.A.M. prepared samples; R.A.M. and L.S.B. performed biochemical and FTIR characterization; V.L. and S.W. collected SSNMR spectroscopy data; S.W. processed and analyzed the data, and calculated the structures; T.O. and A.W. synthesized isotopically labeled retinal; S.-Y.K. and K.-H.J. produced ASR mutants; L.S.B. and V.L. designed the study; S.W., L.S.B. and V.L. wrote the paper. All authors discussed the results of the study.

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Correspondence to Leonid S Brown or Vladimir Ladizhansky.

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Wang, S., Munro, R., Shi, L. et al. Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein. Nat Methods 10, 1007–1012 (2013). https://doi.org/10.1038/nmeth.2635

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