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)
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.
KeywordsAtaxia 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.
- 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. https://doi.org/10.1074/jbc.M501116200 CrossRefGoogle Scholar
- 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. https://doi.org/10.1021/ja00053a051 CrossRefGoogle Scholar
- 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. https://doi.org/10.1021/bi00445a003 CrossRefGoogle Scholar
- 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. https://doi.org/10.1074/jbc.M113.467233 CrossRefGoogle Scholar
- 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. https://doi.org/10.1021/jp501533d CrossRefGoogle Scholar