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)

Abstract

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 H,N, and C 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.

NMR– and MD simulation–based structural characterization of the membrane-associating FATC domain of ataxia telangiectasia mutated

“…Uniformly 15 N-or 15 N- 13 C-labeled proteins were prepared in M9 minimal medium containing 15 NH 4 Cl and/or [ 13 C]glucose as the sole nitrogen and carbon sources. The expression and the purification by a heating step (66) and IgG affinity chromatography were done as described previously (32,67).…”

“…Cells expressing mutant hATMfatc-gb1ent were lysed by sonication in 50 mM Tris, 150 mM NaCl, 2 mM benzamidine, and 0.3 mM EDTA, pH 7.6. Mutant proteins were purified by IgG affinity chromatography as described for the WT and in the manufacturer's manual (32,67). Fractions containing the target protein were pooled, washed with NMR buffer (50 mM Tris, 100 mM NaCl, pH 7.4, for the single mutants F93A ϭ F3049A, W96A ϭ W3052A or pH 6.5 for the double mutant F93A/W96A ϭ F3049A/W3052A), and concentrated.…”

“…Data were processed with NMRPipe (71) and analyzed using NMRView (72). Assignments for the 13 C, 15 N, and 1 H nuclei of micelle-immersed hATMfatc fusion protein were based on 2D 1 H-15 N and various 1 H-13 C HSQC spectra, partially without decoupling, 3D HNCA, HNCACB, CCONH-TOCSY, HCCH-TOCSY, HNHA, HNHB, and 15 N-and 13 Cedited NOESY spectra as described (67). The NOESY mixing times were 90 ms for the 15 N-edited NOESY and 100 ms for the aromatic and aliphatic 13 C-edited NOESY experiments.…”