Biomolecular NMR Assignments

, Volume 12, Issue 1, pp 149–154 | Cite as

1H, 15N, and 13C chemical shift assignments of the micelle immersed FAT C-terminal (FATC) domains of the human protein kinases ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) fused to the B1 domain of streptococcal protein G (GB1)

  • Munirah S. Abd Rahim
  • Lisa A. M. Sommer
  • Anja Wacker
  • Martin Schaad
  • Sonja A. Dames


FAT C-terminal (FATC) is a circa 33 residue-long domain. It controls the kinase functionality in phosphatidylinositol-3 kinase-related kinases (PIKKs). Recent NMR- and CD-monitored interaction studies indicated that the FATC domains of all PIKKs can interact with membrane mimetics albeit with different preferences for membrane properties such as surface charge and curvature. Thus they may generally act as membrane anchoring unit. Here, we present the 1H, 15N, and 13C chemical shift assignments of the DPC micelle immersed FATC domains of the human PIKKs ataxia-telangiectasia mutated (ATM, residues 3024–3056) and DNA protein kinase catalytic subunit (DNA-PKcs, residues 4096–4128), both fused to the 56 residue long B1 domain of Streptococcal protein G (GB1). Each fusion protein is 100 amino acids long and contains in the linking region between the GB1 tag and the FATC region a thrombin (LVPRGS) and an enterokinase (DDDDK) protease site. The assignments pave the route for the detailed structural characterization of the membrane mimetic bound states, which will help to better understand the role of the proper cellular localization at membranes for the function and regulation of PIKKs. The chemical shift assignment of the GB1 tag is useful for NMR spectroscopists developing new experiments or using GB1 otherwise for case studies in the field of in-cell NMR spectroscopy or protein folding. Moreover it is often used as purification tag. Earlier we showed already that GB1 does not interact with membrane mimetics and thus does not disturb the NMR monitoring of membrane mimetic interactions of attached proteins.


Ataxia telangiectasia mutated (ATM) DNA-dependent kinase catalytic subunit (DNA-PKcs) FATC Phosphatidylinositol-3 kinase-related kinases (PIKKs) B1 domain of Streptococcal protein G (GB1) Chemical shift assignment 



This work was supported by a grant from the German Research Foundation to S.A.D. (DA1183/3-1 and -2). S.A.D acknowledges further financial support from the Technische Universität München diversity and talent management office (Laura Bassi award) and the Helmholtz portfolio theme ‘metabolic dysfunction and common disease’ of the Helmholtz Zentrum München. M.S.A.R. is supported by a Ph. D. fellowship from the German academic exchange service (DAAD). Prof. Dr. Michael Sattler and Prof. Dr. Bernd Reif from the Technische Universität München (TUM) and the Helmholtz Zentrum München we thank very much for hosting our group and for sharing their facilities with us.


  1. Archer SJ, Ikura M, Torchia DA, Bax A (1991) An alternative 3D NMR technique for correlating backbone 15N with side chain Hβ resonances in larger proteins. J Magn Reson (1969) 95:636–641. CrossRefGoogle Scholar
  2. Bax A, Clore GM, Gronenborn AM (1990) 1H–1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J Magn Reson 88:425–431. ADSGoogle Scholar
  3. Bosotti R, Isacchi A, Sonnhammer EL (2000) FAT: a novel domain in PIK-related kinases. Trends Biochem Sci 25:225–227CrossRefGoogle Scholar
  4. Byeon IJ, Louis JM, Gronenborn AM (2004) A captured folding intermediate involved in dimerization and domain-swapping of GB1. J Mol Biol 340:615–625. CrossRefGoogle Scholar
  5. Chen BP, Li M, Asaithamby A (2012) New insights into the roles of ATM and DNA-PKcs in the cellular response to oxidative stress. Cancer Lett 327:103–110. CrossRefGoogle Scholar
  6. Cheng Y, Patel DJ (2004) An efficient system for small protein expression and refolding. Biochem Biophys Res Commun 317:401–405. CrossRefGoogle Scholar
  7. Clore GM, Gronenborn AM, Bax A (1998) A robust method for determining the magnitude of the fully asymmetric alignment tensor of oriented macromolecules in the absence of structural information. J Magn Reson (San Diego, CA: 1997) 133:216–221. Google Scholar
  8. Dames SA (2010) Structural basis for the association of the redox-sensitive target of rapamycin FATC domain with membrane-mimetic micelles. J Biol Chem 285:7766–7775. CrossRefGoogle Scholar
  9. Dames SA, Mulet JM, Rathgeb-Szabo K, Hall MN, Grzesiek S (2005) The solution structure of the FATC domain of the protein kinase target of rapamycin suggests a role for redox-dependent structural and cellular stability. J Biol Chem 280:20558–20564. CrossRefGoogle Scholar
  10. De Cicco M, Rahim MS, Dames SA (2015) Regulation of the target of rapamycin and other phosphatidylinositol-3 kinase-related kinases by membrane targeting. Membranes 5:553–575. CrossRefGoogle Scholar
  11. 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
  12. Ding K, Louis JM, Gronenborn AM (2004) Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR. J Mol Biol 335:1299–1307CrossRefGoogle Scholar
  13. Frueh DP, Arthanari H, Wagner G (2005) Unambiguous assignment of NMR protein backbone signals with a time-shared triple-resonance experiment. J Biomol NMR 33:187–196. CrossRefGoogle Scholar
  14. Grzesiek S, Bax A (1992) Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. J Magn Reson (1969) 96:432–440. CrossRefGoogle Scholar
  15. Grzesiek S, Anglister J, Bax A (1993) Correlation of backbone amide and aliphatic side-chain resonances in 13C/15N-enriched proteins by isotropic mixing of 13C magnetization. J Magn Reson Ser B 101:114–119. CrossRefGoogle Scholar
  16. Hoke SM et al (2010) Mutational analysis of the C-terminal FATC domain of saccharomyces cerevisiae Tra1. Curr Genet 56:447–465CrossRefGoogle Scholar
  17. Huang A, de Jong RN, Folkers GE, Boelens R (2010) NMR characterization of foldedness for the production of E3 RING domains. J Struct Biol 172:120–127. CrossRefGoogle Scholar
  18. Huth JR, Bewley CA, Clore GM, Gronenborn AM, Jackson BM, Hinnebusch AG (1997) Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR. Protein Sci 6:2359–2364. CrossRefGoogle Scholar
  19. Jiang X, Sun Y, Chen S, Roy K, Price BD (2006) The FATC domains of PIKK proteins are functionally equivalent and participate in the Tip60-dependent activation of DNA-PKcs and ATM. J Biol Chem 281:15741–15746. CrossRefGoogle Scholar
  20. Johnson BA (2004) Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol Biol 278:313–352. Google Scholar
  21. Koenig BW, Rogowski M, Louis JM (2003) A rapid method to attain isotope labeled small soluble peptides for NMR studies. J Biomol NMR 26:193–202CrossRefGoogle Scholar
  22. Kong X, Shen Y, Jiang N, Fei X, Mi J (2011) Emerging roles of DNA-PK besides DNA repair. Cell Signal 23:1273–1280. CrossRefGoogle Scholar
  23. Kruger A, Ralser M (2011) ATM is a redox sensor linking genome stability and carbon metabolism. Sci Signal 4:pe17. CrossRefGoogle Scholar
  24. Lempiäinen H, Halazonetis TD (2009) Emerging common themes in regulation of PIKKs and PI3Ks. EMBO J 28:3067–3073. CrossRefGoogle Scholar
  25. Lovejoy CA, Cortez D (2009) Common mechanisms of PIKK regulation. DNA Repair 8:1004–1008. CrossRefGoogle Scholar
  26. Luchinat E, Banci L (2016) A unique tool for cellular structural biology: in-cell NMR. J Biol Chem 291:3776–3784. CrossRefGoogle Scholar
  27. Lyons BA, Montelione GT (1993) An HCCNH triple-resonance experiment using carbon-13 isotropic mixing for correlating backbone amide and side-chain aliphatic resonances in isotopically enriched proteins. J Magn Reson Ser B 101:206–209. CrossRefGoogle Scholar
  28. Montelione GT, Lyons BA, Emerson SD, Tashiro M (1992) An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically-enriched proteins. J Am Chem Soc 114:10974–10975. CrossRefGoogle Scholar
  29. Mordes DA, Glick GG, Zhao R, Cortez D (2008) TopBP1 activates ATR through ATRIP and a PIKK regulatory domain. Genes Dev 22:1478–1489. CrossRefGoogle Scholar
  30. Morita T, Yamashita A, Kashima I, Ogata K, Ishiura S, Ohno S (2007) Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay. J Biol Chem 282:7799–7808. CrossRefGoogle Scholar
  31. Neri D, Szyperski T, Otting G, Senn H, Wuethrich K (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional carbon-13 labeling. Biochemistry 28:7510–7516. CrossRefGoogle Scholar
  32. Olejniczak ET, Xu RX, Fesik SW (1992) A 4D HCCH-TOCSY experiment for assigning the side chain1H and13C resonances of proteins. J Biomol NMR 2:655–659. CrossRefGoogle Scholar
  33. Perry J, Kleckner N (2003) The ATRs, ATMs, and TORs are giant HEAT repeat proteins. Cell 112:151–155CrossRefGoogle Scholar
  34. Selenko P, Serber Z, Gadea B, Ruderman J, Wagner G (2006) Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes. Proc Natl Acad Sci USA 103:11904–11909. ADSCrossRefGoogle Scholar
  35. Shiloh Y (2003) ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 3:155–168. CrossRefGoogle Scholar
  36. Shiloh Y, Ziv Y (2013) The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14:197–210CrossRefGoogle Scholar
  37. Sommer LAM, Dames SA (2014) Characterization of residue-dependent differences in the peripheral membrane association of the FATC domain of the kinase ‘target of rapamycin’ by NMR and CD spectroscopy. FEBS Lett 588:1755–1766. CrossRefGoogle Scholar
  38. Sommer LAM, Meier MA, Dames SA (2012) A fast and simple method for probing the interaction of peptides and proteins with lipids and membrane-mimetics using GB1 fusion proteins and NMR spectroscopy. Protein Sci 21:1566–1570. CrossRefGoogle Scholar
  39. Sommer LAM, Schaad M, Dames SA (2013) NMR- and circular dichroism-monitored lipid binding studies suggest a general role for the FATC domain as membrane anchor of phosphatidylinositol-3 kinase-related kinases (PIKK). J Biol Chem 288:20046–20063. CrossRefGoogle Scholar
  40. Sommer LAM, Janke JJ, Bennett WFD, Bürck J, Ulrich AS, Tieleman DP, Dames SA (2014) Characterization of the immersion properties of the peripheral membrane anchor of the FATC domain of the kinase “target of rapamycin” by NMR, oriented CD spectroscopy, and MD simulations. J Phys Chem B 118:4817–4831. CrossRefGoogle Scholar
  41. Vuister GW, Bax A (1993) Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNH.alpha.) coupling constants in 15N-enriched proteins. J Am Chem Soc 115:7772–7777. CrossRefGoogle Scholar
  42. Vuister GW, Bax A (1994) Measurement of four-bond HN–Hα J-couplings in staphylococcal nuclease. J Biomol NMR 4:193–200. CrossRefGoogle Scholar
  43. Walsh JD, Meier K, Ishima R, Gronenborn AM (2010) NMR studies on domain diffusion and alignment in modular GB1 repeats. Biophys J 99:2636–2646CrossRefGoogle Scholar
  44. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484. CrossRefGoogle Scholar

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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Chemistry, Biomolecular NMR SpectroscopyTechnische Universität MünchenGarchingGermany
  2. 2.Roche Diagnostics GmbHCentralised and Point of Care SolutionsPenzbergGermany
  3. 3.Division of Radiopharmaceutical ChemistryGerman Cancer Research Center (DKFZ)HeidelbergGermany
  4. 4.Quintiles AGBaselSwitzerland
  5. 5.Institute of Structural BiologyHelmholtz Zentrum MünchenNeuherbergGermany

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