Biomolecular NMR Assignments

, Volume 10, Issue 1, pp 79–83 | Cite as

Backbone and side-chain resonance assignment of the A147T polymorph of mouse TSPO in complex with a high-affinity radioligand

  • Mariusz Jaremko
  • Łukasz Jaremko
  • Karin Giller
  • Stefan Becker
  • Markus ZweckstetterEmail author


The integral polytopic membrane protein TSPO is the target for numerous endogenous and synthetic ligands. However, the affinity of many ligands is influenced by a common polymorphism in TSPO, in which an alanine at position 147 is replaced by threonine, thereby complicating the use of several radioligands for clinical diagnosis. In contrast, the best-characterized TSPO ligand (R)-PK11195 binds with similar affinity to both variants of mitochondrial TSPO (wild-type and A147T variant). Here we report the 1H, 13C, 15N backbone and side-chain resonance assignment of the A147T polymorph of TSPO from Mus Musculus in complex with (R)-PK11195 in DPC detergent micelles. More than 90 % of all resonances were sequence-specifically assigned, demonstrating the ability to obtain high-quality spectral data for both the backbone and the side-chains of medically relevant integral membrane proteins.


TSPO Polymorphism (R)-PK11195 NMR spectroscopy Membrane protein 



1H, 13C, 15N resonance assignments of the A147T-mTSPO/PK11195 complex have been deposited at the BMRB data base (Accession Code: 25513). This work was partially supported by the DFG Collaborative Research Center 803, Project A11.

Compliance with ethical standards

Conflict of interest

No conflict declared.


  1. Banati RB, Middleton RJ, Chan R, Hatty CR, Kam WWY, Quin C, Graeber MB, Parmar A, Zahra D, Callaghan P, Fok S, Howell NR, Gregoire M, Szabo A, Pham T, Davis E, Liu GJ (2014) Positron emission tomography and functional characterization of a complete PBR/TSPO knockout. Nat Commun 5:5452. doi: 10.1038/ncomms6452 ADSCrossRefGoogle Scholar
  2. Bax A, Clore GM, Gronenborn AM (1990) 1H–1H correlation via isotropic mixing of 13C magnetization, a new 3-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J Magn Reson 88:425–431. doi: 10.1007/BF00197809 ADSGoogle Scholar
  3. Fan J, Lindemann P, Feuilloley MG, Papadopoulos V (2012) Structural and functional evolution of the translocator protein (18 kDa). Curr Mol Med 12:369–386. doi: 10.2174/1566524011207040369 Google Scholar
  4. Gatliff J, Campanella M (2012) The 18 kDa translocator protein (TSPO): a new perspective in mitochondrial biology. Curr Mol Med 12:356–368. doi: 10.2174/1566524011207040356 Google Scholar
  5. Girard C, Liu S, Adams D, Lacoix C, Sinéus M, Boucher C, Papadopoulos V, Rupprecht R, Schumacher M, Groyer G (2012) Axonal regeneration and neuroinflammation: roles for the translocator protein 18 kDa. J Neuroendocrinol 24(1):71–81. doi: 10.1111/j.1365-2826.2011.02215.x CrossRefGoogle Scholar
  6. Guo Y, Kalathur RC, Liu Q, Kloss B, Bruni R, Ginter C, Kloppmann E, Rost B, Hendrickson WA (2015) Protein structure. Structure and activity of tryptophan-rich TSPO proteins. Science 347:551–555. doi: 10.1126/science.aaa1534 ADSCrossRefGoogle Scholar
  7. Jamin N, Neumann JM, Ostuni MA, Vu TK, Yao ZX, Murail S, Robert JC, Giatzakis C, Papadopoulos V, Lacapère JJ (2005) Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor. Mol Endocrinol 19:588–594. doi: 10.1210/me.2004-0308 CrossRefGoogle Scholar
  8. Jaremko L, Jaremko M, Giller K, Becker S, Zweckstetter M (2014) Structure of the mitochondrial translocator protein in complex with a diagnostic ligand. Science 343:1363–1366. doi: 10.1126/science.1248725 ADSCrossRefGoogle Scholar
  9. Jaremko M, Jaremko L, Giller K, Becker S, Zweckstetter M (2015) Structural integrity of the A147T polymorph of mammalian TSPO. ChemBioChem. doi: 10.1002/cbic.201500217 Google Scholar
  10. Keller RLJ (2004) The computer aided resonance assignment tutorial. Cantina, GoldauGoogle Scholar
  11. Kugler W, Veenman L, Shandalov Y, Leschiner S, Spanier I, Lakomek M, Gavish M (2008) Ligands of the mitochondrial 18 kDa translocator protein attenuate apoptosis of human glioblastoma cells exposed to erucylphosphohomocholine. Cell Oncol 30:435–450Google Scholar
  12. Lacapère JJ, Delavoie F, Li H, Péranzi G, Maccario J, Papadopoulos V, Vidic B (2001) Structural and functional study of reconstituted peripheral benzodiazepine receptor. Biochem Biophys Res Commun 284:536–541. doi: 10.1006/bbrc.2001.4975 CrossRefGoogle Scholar
  13. Li F, Liu J, Zheng Y, Garavito RM, Ferguson-Miller S (2015) Protein structure. Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism. Science 347:555–558. doi: 10.1126/science.1260590 ADSCrossRefGoogle Scholar
  14. Liu GJ, Middleton RJ, Hatty CR, Kam WW, Chan R, Pham T, Harrison-Brown M, Dodson E, Veale K, Banati RB (2014) The 18 kDa translocator protein, microglia and neuroinflammation. Brain Pathol 24:631–653. doi: 10.1111/bpa.12196 CrossRefGoogle Scholar
  15. Marion D, Driscoll PC, Kay LE, Wingfield PT, Bax A, Gronenborn AM, Clore GM (1989) Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H–15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1β. Biochemistry 28:6150–6156. doi: 10.1186/s12977-014-0094-8 CrossRefGoogle Scholar
  16. Midzak A, Akula N, Lecanu L, Papadopoulos V (2011) Novel androstenetriol interacts with the mitochondrial translocator protein and controls steroidogenesis. J Biol Chem 286(11):9875–9887. doi: 10.1074/jbc.M110.203216 CrossRefGoogle Scholar
  17. Murail S, Robert JC, Coïc YM, Neumann JM, Ostuni MA, Yao ZX, Papadopoulos V, Jamin N, Lacapère JJ (2008) Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding. Biochim Biophys Acta 1778:1375–1381. doi: 10.1016/j.bbamem.2008.03.012 CrossRefGoogle Scholar
  18. Owen DR, Lewis AJ, Reynolds R, Rupprecht R, Eser D, Wilkins MR, Bennacef I, Nutt DJ, Parker CA (2011) Variation in binding affinity of the novel anxiolytic XBD173 for the 18 kDa translocator protein in human brain. Synapse 65:257–259. doi: 10.1002/syn.20884 CrossRefGoogle Scholar
  19. Owen DR, Yeo AJ, Gunn RN, Song K, Wadsworth G, Lewis A, Rhodes C, Pulford DJ, Bennacef I, Parker CA, StJean PL, Cardon LR, Mooser VE, Matthews PM, Rabiner EA, Rubio JP (2012) An 18 kDa translocator protein (TSPO) polymorphism explains differences in binding affinity of the PET radioligand PBR28. J Cereb Blood Flow Metab 32:1–5. doi: 10.1038/jcbfm.2011.147 CrossRefGoogle Scholar
  20. Papadopoulos V, Amri H, Boujrad N, Cascio C, Culty M, Garnier M, Hardwick M, Li H, Vidic B, Brown AS, Reversa JL, Bernassau JM, Drieu K (1997) Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62:21–28. doi: 10.1016/S0039-128X(96)00154-7 CrossRefGoogle Scholar
  21. Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapère JJ, Lindemann P, Norenberg MD, Nutt D, Weizman A, Zhang MR, Gavish M (2006) Translocator protein (18 kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol Sci 27(8):402–409CrossRefGoogle Scholar
  22. Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371. doi: 10.1073/pnas.94.23.12366 ADSCrossRefGoogle Scholar
  23. Salzmann M, Pervushin K, Wider G, Senn H, Wüthrich K (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci USA 95:13585–13590. doi: 10.1073/pnas.95.23.13585 ADSCrossRefGoogle Scholar
  24. Scarf AM, Luus C, Da Pozzo E, Selleri S, Guarino C, Martini C, Ittner LM, Kassiou M (2012) Evidence for complex binding profiles and species differences at the translocator protein (TSPO) (18 kDa). Curr Mol Med 12:488–493. doi: 10.2174/1566524011207040488 Google Scholar
  25. Shen Y, Bax A (2010) Prediction of Xaa-Pro peptide bond conformation from sequence and chemical shifts. J Biomol NMR 46:199–204. doi: 10.1007/s10858-009-9395-y CrossRefGoogle Scholar
  26. Stocco DM (2014) The role of PBR/TSPO in steroid biosynthesis challenged. Endocrinology 155:6–9. doi: 10.1210/en.2013-2041 CrossRefGoogle Scholar
  27. Taliani S, Da Settimo F, Da Pozzo E, Chelli B, Martini C (2009) Translocator protein ligands as promising therapeutic tools for anxiety disorders. Curr Med Chem 16:3359–3380. doi: 10.2174/092986709789057653 CrossRefGoogle Scholar
  28. Tu LN, Morohaku K, Manna PR, Pelton SH, Butler WR, Stocco DM, Selvaraj V (2014) Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis. J Biol Chem 289:27444–27454. doi: 10.1074/jbc.M114.578286 CrossRefGoogle Scholar
  29. Tu LN, Zhao AH, Stocco DM, Selvaraj V (2015) PK11195 effect on steroidogenesis is not mediated through the translocator protein (TSPO). Endocrinology 156:1033–1039. doi: 10.1210/en.2014-1707 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Mariusz Jaremko
    • 1
  • Łukasz Jaremko
    • 1
    • 2
  • Karin Giller
    • 1
  • Stefan Becker
    • 1
  • Markus Zweckstetter
    • 1
    • 2
    • 3
    Email author
  1. 1.Max-Planck-Institut für Biophysikalische ChemieGöttingenGermany
  2. 2.Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE)GöttingenGermany
  3. 3.Center for Nanoscale Microscopy and Molecular Physiology of the BrainUniversity Medical CenterGöttingenGermany

Personalised recommendations