Journal of Biomolecular NMR

, Volume 73, Issue 6–7, pp 347–364 | Cite as

A unified structural model of the mammalian translocator protein (TSPO)

  • Yan Xia
  • Kaitlyn Ledwitch
  • Georg Kuenze
  • Amanda Duran
  • Jun Li
  • Charles R. Sanders
  • Charles Manning
  • Jens MeilerEmail author


The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a membrane protein located on the outer mitochondrial membrane. Experimentally-derived structures of mouse TSPO (mTSPO) and its homologs from bacterial species have been determined by NMR spectroscopy and X-ray crystallography, respectively. These structures and ligand interactions within the TSPO binding pocket display distinct differences. Here, we leverage experimental and computational studies to derive a unified structural model of mTSPO in the presence and absence of the TSPO ligand, PK11195, and study the effects of DPC detergent micelles on the TSPO structure and ligand binding. From this work, we conclude that that the lipid-mimetic system used to solubilize mTSPO for NMR studies thermodynamically destabilizes the protein, introduces structural perturbations, and alters the characteristics of ligand binding. Furthermore, we used Rosetta to construct a unified mTSPO model that reconciles deviating features of the mammalian and bacterial TSPO. These deviating features are likely a consequence of the detergent system used for structure determination of mTSPO by NMR. The unified mTSPO model agrees with available experimental NMR data, appears to be physically realistic (i.e. thermodynamically not frustrated as judged by the Rosetta energy function), and simultaneously shares the structural features observed in sequence-conserved regions of the bacterial proteins. Finally, we identified the binding site for an imaging ligand VUIIS8310 that is currently positioned for clinical translation using NMR spectroscopy and propose a computational model of the VUIIS8310-mTSPO complex.


Translocator protein (TSPO) NMR spectroscopy Rosetta Homology modeling Protein folding Ligand docking 



This work was conducted using the computing cluster of the Advanced Center for Research and Education (ACCRE) at Vanderbilt University, TN. Work in the Meiler laboratory is supported through NIH (R01 GM080403, R01 GM099842) and NSF (CHE 1305874). CRS was supported by NIH RF1 AG056147. VUIIS8310 was developed with support from NIH R01 CA163806 (HCM) and the Kleberg Foundation (HCM). The authors would also like to thank Dr. Markus Voehler and the Biomolecular NMR Facility at Vanderbilt for support and advice regarding the NMR data acquisition.

Supplementary material

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Supplementary material 1 (DOCX 4206 kb)


  1. Banati RB, Middleton RJ, Chan R, Hatty CR, Kam WW, 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 5:5452ADSCrossRefGoogle Scholar
  2. Barth P, Schonbrun J, Baker D (2007) Toward high-resolution prediction and design of transmembrane helical protein structures. Proc Natl Acad Sci USA 104:15682–15687ADSCrossRefGoogle Scholar
  3. Batarseh A, Papadopoulos V (2010) Regulation of translocator protein 18 kDa (TSPO) expression in health and disease states. Mol Cell Endocrinol 327:1–12CrossRefGoogle Scholar
  4. Black KL, Ikezaki K, Toga AW (1989) Imaging of brain tumors using peripheral benzodiazepine receptor ligands. J Neurosurg 71:113–118CrossRefGoogle Scholar
  5. Braestrup C, Squires RF (1977) Specific benzodiazepine receptors in rat brain characterized by high-affinity (3H)diazepam binding. Proc Natl Acad Sci USA 74:3805–3809ADSCrossRefGoogle Scholar
  6. Carmel I, Fares FA, Leschiner S, Scherubl H, Weisinger G, Gavish M (1999) Peripheral-type benzodiazepine receptors in the regulation of proliferation of MCF-7 human breast carcinoma cell line. Biochem Pharmacol 58:273–278CrossRefGoogle Scholar
  7. Cheung Y-Y, Buck J, Nickels M, Tang D, Manning HC (2014) Preclinical evaluation of 7-chloro-N, N,5-trimethyl-4-oxo-3(6-[18F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide: a novel pyridazinoindole ligand for PET imaging of TSPO in cancer. J Nucl Med 55:1053Google Scholar
  8. Ching AS, Kuhnast B, Damont A, Roeda D, Tavitian B, Dolle F (2012) Current paradigm of the 18-kDa translocator protein (TSPO) as a molecular target for PET imaging in neuroinflammation and neurodegenerative diseases. Insights Imaging 3:111–119CrossRefGoogle Scholar
  9. Cleary J, Johnson KM, Opipari AW Jr, Glick GD (2007) Inhibition of the mitochondrial F1F0-ATPase by ligands of the peripheral benzodiazepine receptor. Bioorg Med Chem Lett 17:1667–1670CrossRefGoogle Scholar
  10. Conway P, Tyka MD, DiMaio F, Konerding DE, Baker D (2014) Relaxation of backbone bond geometry improves protein energy landscape modeling. Protein Sci 23:47–55CrossRefGoogle Scholar
  11. Cross TA, Sharma M, Yi M, Zhou H-X (2011) Influence of solubilizing environments on membrane protein structures. Trends Biochem Sci 36:117–125CrossRefGoogle Scholar
  12. Cross TA, Murray DT, Watts A (2013) Helical membrane protein conformations and their environment. Eur Biophys J 42:731–755CrossRefGoogle Scholar
  13. Deane NG, Manning HC, Foutch AC, Washington MK, Aronow BJ, Bornhop DJ, Coffey RJ (2007) Targeted imaging of colonic tumors in smad3-/- mice discriminates cancer and inflammation. Mol Cancer Res 5:341–349CrossRefGoogle Scholar
  14. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  15. Fafalios A, Akhavan A, Parwani AV, Bies RR, McHugh KJ, Pflug BR (2009) Translocator protein blockade reduces prostate tumor growth. Clin Cancer Res 15:6177–6184CrossRefGoogle Scholar
  16. Fan J, Lindemann P, Feuilloley MG, Papadopoulos V (2012) Structural and functional evolution of the translocator protein (18 kDa). Curr Mol Med 12:369–386Google Scholar
  17. Frison M, Mallach AK, Kennedy E, Campanella M (2017) The 18 kDa Translocator protein (TSPO): cholesterol trafficking and the biology of a prognostic and therapeutic mitochondrial target. In: Rostovtseva TK (ed) Molecular basis for mitochondrial signaling. Springer, Cham, pp 285–315CrossRefGoogle Scholar
  18. Galiegue S, Tinel N, Casellas P (2003) The peripheral benzodiazepine receptor: a promising therapeutic drug target. Curr Med Chem 10:1563–1572CrossRefGoogle Scholar
  19. Gavish M, Bachman I, Shoukrun R, Katz Y, Veenman L, Weisinger G, Weizman A (1999) Enigma of the peripheral benzodiazepine receptor. Pharmacol Rev 51:629–650Google Scholar
  20. Ginter C, Kiburu I, Boudker O (2013) Chemical catalysis by the translocator protein (18 kDa). Biochemistry 52:3609–3611CrossRefGoogle Scholar
  21. Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte M, Glick GD, Petronilli V, Zoratti M, Szabo I, Lippe G, Bernardi P (2013) Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci USA 110:5887–5892ADSCrossRefGoogle Scholar
  22. Gregory KJ, Nguyen ED, Reiff SD, Squire EF, Stauffer SR, Lindsley CW, Meiler J, Conn PJ (2013) Probing the metabotropic glutamate receptor 5 (mGlu(5)) positive allosteric modulator (PAM) binding pocket: discovery of point mutations that engender a “molecular switch” in PAM pharmacology. Mol Pharmacol 83:991–1006CrossRefGoogle Scholar
  23. 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 347:551–555ADSCrossRefGoogle Scholar
  24. Han Z, Slack RS, Li W, Papadopoulos V (2003) Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression. J Recept Signal Transduct Res 23:225–238CrossRefGoogle Scholar
  25. Hardwick M, Fertikh D, Culty M, Li H, Vidic B, Papadopoulos V (1999) Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res 59:831–842Google Scholar
  26. Hardwick M, Rone J, Han ZQ, Haddad B, Papadopoulos V (2001) Peripheral-type benzodiazepine receptor levels correlate with the ability of human breast cancer MDA-MB-231 cell line to grow in SCID mice. Int J Cancer 94:322–327CrossRefGoogle Scholar
  27. Hatty CR, Le Brun AP, Lake V, Clifton LA, Liu GJ, James M, Banati RB (2014) Investigating the interactions of the 18 kDa translocator protein and its ligand PK11195 in planar lipid bilayers. Biochim Biophys Acta 1838:1019–1030CrossRefGoogle Scholar
  28. Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549CrossRefGoogle Scholar
  29. Jaipuria G, Leonov A, Giller K, Vasa SK, Jaremko L, Jaremko M, Linser R, Becker S, Zweckstetter M (2017) Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure. Nat Commun 8:14893ADSCrossRefGoogle Scholar
  30. Jamin N, Neumann JM, Ostuni MA, Vu TK, Yao ZX, Murail S, Robert JC, Giatzakis C, Papadopoulos V, Lacapere JJ (2005) Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor. Mol Endocrinol 19:588–594CrossRefGoogle Scholar
  31. Jaremko Ł, Jaremko M, Giller K, Becker S, Zweckstetter M (2014) Structure of the mitochondrial translocator protein in complex with a diagnostic ligand. Science 343:1363–1366ADSCrossRefGoogle Scholar
  32. Jaremko M, Jaremko L, Giller K, Becker S, Zweckstetter M (2015a) Structural integrity of the A147T polymorph of mammalian TSPO. ChemBioChem 16:1483–1489CrossRefGoogle Scholar
  33. Jaremko L, Jaremko M, Giller K, Becker S, Zweckstetter M (2015b) Conformational flexibility in the transmembrane protein TSPO. Chemistry 21:16555–16563CrossRefGoogle Scholar
  34. Junck L, Olson JMM, Ciliax BJ, Koeppe RA, Watkins GL, Jewett DM, Mckeever PE, Wieland DM, Kilbourn MR, Starostarubinstein S, Mancini WR, Kuhl DE, Greenberg HS, Young AB (1989) Pet imaging of human gliomas with ligands for the peripheral benzodiazepine binding-site. Ann Neurol 26:752–758CrossRefGoogle Scholar
  35. Korkhov VM, Sachse C, Short JM, Tate CG (2010) Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals. Structure 18:677–687CrossRefGoogle Scholar
  36. Kreisl WC, Jenko KJ, Hines CS, Lyoo CH, Corona W, Morse CL, Zoghbi SS, Hyde T, Kleinman JE, Pike VW, McMahon FJ, Innis RB (2013) A genetic polymorphism for translocator protein 18 kDa affects both in vitro and in vivo radioligand binding in human brain to this putative biomarker of neuroinflammation. J Cereb Blood Flow Metab 33:53–58CrossRefGoogle Scholar
  37. Lacapère J-J, Papadopoulos V (2003) Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis. Steroids 68:569–585CrossRefGoogle Scholar
  38. Lacapere JJ, Delavoie F, Li H, Peranzi G, Maccario J, Papadopoulos V, Vidic B (2001) Structural and functional study of reconstituted peripheral benzodiazepine receptor. Biochem Biophys Res Commun 284:536–541CrossRefGoogle Scholar
  39. Lacapere J-J, Iatmanen-Harbi S, Senicourt L, Lequin O, Tekely P, Purusottam RN, Hellwig P, Kriegel S, Ravaud S, Juillan-Binard C (2014) Structural studies of TSPO, a mitochondrial membrane protein. In: Mus-Veteau I (ed) Membrane proteins production for structural analysis. Springer, New York, pp 393–421Google Scholar
  40. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefGoogle Scholar
  41. Lazaridis T (2003) Effective energy function for proteins in lipid membranes. Proteins 52:176–192CrossRefGoogle Scholar
  42. Lazaridis T, Karplus M (1999) Effective energy function for proteins in solution. Proteins 35:133–152CrossRefGoogle Scholar
  43. Lee W, Tonelli M, Markley JL (2015) NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics (Oxford, England) 31:1325–1327CrossRefGoogle Scholar
  44. Li H, Papadopoulos V (1998) Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus patterns. Endocrinology 139:4991–4997CrossRefGoogle Scholar
  45. 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 347:555–558ADSCrossRefGoogle Scholar
  46. 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 24:631–653CrossRefGoogle Scholar
  47. Maaser K, Hèopfner M, Jansen A, Weisinger G, Gavish M, Kozikowski AP, Weizman A, Carayon P, Riecken EO, Zeitz M, Scherèubl H, Medical Clinic I (2001) Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells. Br. J Cancer 85(11):1771–1780CrossRefGoogle Scholar
  48. Maaser K, Grabowski P, Sutter AP, Hopfner M, Foss HD, Stein H, Berger G, Gavish M, Zeitz M, Scherubl H (2002) Overexpression of the peripheral benzodiazepine receptor is a relevant prognostic factor in stage III colorectal cancer. Clin Cancer Res 8:3205–3209Google Scholar
  49. McEnery MW, Snowman AM, Trifiletti RR, Snyder SH (1992) Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc Natl Acad Sci USA 89:3170–3174ADSCrossRefGoogle Scholar
  50. Meiler J, Baker D (2006) ROSETTALIGAND: protein-small molecule docking with full side-chain flexibility. Proteins 65:538–548CrossRefGoogle Scholar
  51. Molecular Operating Environment, 2013.08; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2013Google Scholar
  52. Morohaku K, Pelton SH, Daugherty DJ, Butler WR, Deng W, Selvaraj V (2014) Translocator protein/peripheral benzodiazepine receptor is not required for steroid hormone biosynthesis. Endocrinology 155:89–97CrossRefGoogle Scholar
  53. Murail S, Robert JC, Coic YM, Neumann JM, Ostuni MA, Yao ZX, Papadopoulos V, Jamin N, Lacapere 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 1778:1375–1381CrossRefGoogle Scholar
  54. 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 32:1–5CrossRefGoogle Scholar
  55. Papadopoulos V, Boujrad N, Ikonomovic MD, Ferrara P, Vidic B (1994) Topography of the Leydig cell mitochondrial peripheral-type benzodiazepine receptor. Mol Cell Endocrinol 104:R5–R9CrossRefGoogle Scholar
  56. Schrodinger LLC (2015) The PyMOL Molecular Graphics System, Version 1.8Google Scholar
  57. Shen Y, Bax A (2010) SPARTA + : a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural network. J Biomol NMR 48:13–22CrossRefGoogle Scholar
  58. Sileikyte J, Blachly-Dyson E, Sewell R, Carpi A, Menabo R, Di Lisa F, Ricchelli F, Bernardi Forte M (2014) Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (TSPO). J Biol Chem 289:13769–13781CrossRefGoogle Scholar
  59. Song Y, DiMaio F, Wang RY, Kim D, Miles C, Brunette T, Thompson J, Baker D (2013) High-resolution comparative modeling with RosettaCM. Structure 21:1735–1742CrossRefGoogle Scholar
  60. Starostarubinstein S, Ciliax BJ, Penney JB, Mckeever P, Young AB (1987) Imaging of a glioma using peripheral benzodiazepine receptor ligands. Proc Natl Acad Sci USA 84:891–895ADSCrossRefGoogle Scholar
  61. Taketani S, Kohno H, Furukawa T, Tokunaga R (1995) Involvement of peripheral-type benzodiazepine receptors in the intracellular transport of heme and porphyrins. J Biochem 117:875–880CrossRefGoogle Scholar
  62. Tang D, Hight MR, McKinley ET, Fu A, Buck JR, Smith RA, Tantawy MN, Peterson TE, Colvin DC, Ansari MS, Nickels M, Manning HC (2012) Quantitative preclinical imaging of TSPO expression in glioma using N, N-diethyl-2-(2-(4-(2–18F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimi din-3-yl)acetamide. J Nucl Med 53:287–294CrossRefGoogle Scholar
  63. Tang D, McKinley ET, Hight MR, Uddin MI, Harp JM, Fu A, Nickels ML, Buck JR, Manning HC (2013) Synthesis and structure-activity relationships of 5,6,7-substituted pyrazolopyrimidines: discovery of a novel TSPO PET ligand for cancer imaging. J Med Chem 56:3429–3433CrossRefGoogle Scholar
  64. Tang D, Nickels ML, Tantawy MN, Buck JR, Manning HC (2014) Preclinical imaging evaluation of novel TSPO-PET ligand 2-(5,7-Diethyl-2-(4-(2-[F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N, N- diethylacetamide ([F]VUIIS1008) in Glioma. Mol Imaging Biol 16:813–820CrossRefGoogle Scholar
  65. Tang D, Li J, Buck JR, Tantawy MN, Xia Y, Harp JM, Nickels ML, Meiler J, Manning HC (2016) Evaluation of TSPO PET ligands [18F]VUIIS1009A and [18F]VUIIS1009B: tracers for cancer imaging. Mol Imaging Biol 19:578–588CrossRefGoogle Scholar
  66. Teboul D, Beaufils S, Taveau J-C, Iatmanen-Harbi S, Renault A, Venien-Bryan C, Vie VR, Lacapere J-J (2012) Mouse TSPO in a lipid environment interacting with a functionalized monolayer. Biochim Biophys Acta 1818:2791–2800CrossRefGoogle Scholar
  67. 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 289:27444–27454CrossRefGoogle Scholar
  68. Tu LN, Zhao AH, Stocco DM, Selvaraj V (2015) PK11195 effect on steroidogenesis is not mediated through the translocator protein (TSPO). Endocrinology 156:1033–1039CrossRefGoogle Scholar
  69. Veenman L, Gavish M (2012) The role of 18 kDa mitochondrial translocator protein (TSPO) in programmed cell death, and effects of steroids on TSPO expression. Curr Mol Med 12:398–412Google Scholar
  70. Verma A, Nye JS, Snyder SH (1987) Porphyrins are endogenous ligands for the mitochondrial (peripheral-type) benzodiazepine receptor. Proc Natl Acad Sci USA 84:2256–2260ADSCrossRefGoogle Scholar
  71. Yarov-Yarovoy V, Schonbrun J, Baker D (2006) Multipass membrane protein structure prediction using Rosetta. Proteins 62:1010–1025CrossRefGoogle Scholar
  72. Zhou HX, Cross TA (2013) Influences of membrane mimetic environments on membrane protein structures. Ann Rev Biophys 42:361–392CrossRefGoogle Scholar

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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Center for Structural BiologyVanderbilt UniversityNashvilleUSA
  2. 2.Department of ChemistryVanderbilt UniversityNashvilleUSA
  3. 3.Institute of Imaging ScienceVanderbilt University Medical CenterNashvilleUSA
  4. 4.Department of BiochemistryVanderbilt UniversityNashvilleUSA
  5. 5.Department of Chemistry, Center for Structural BiologyVanderbilt UniversityNashvilleUSA

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