Solution NMR assignment of the ARC4 domain of human tankyrase 2

Abstract

Tankyrases are poly(ADP-ribose)polymerases (PARPs) which recognize their substrates via their ankyrin repeat cluster (ARC) domains. The human tankyrases (TNKS/TNKS2) contain five ARCs in their extensive N-terminal region; of these, four bind peptides present within tankyrase interactors and substrates. These short, linear segments, known as tankyrase-binding motifs (TBMs), contain some highly conserved features: an arginine at position 1, which occupies a predominantly acidic binding site, and a glycine at position 6 that is sandwiched between two aromatic side chains on the surface of the ARC domain. Tankyrases are involved in a multitude of biological functions, amongst them Wnt/β-catenin signaling, the maintenance of telomeres, glucose metabolism, spindle formation, the DNA damage response and Hippo signaling. As many of these are relevant to human disease, tankyrase is an important target candidate for drug development. With the emergence of non-catalytic (scaffolding) functions of tankyrase, it seems attractive to interfere with ARC function rather than the enzymatic activity of tankyrase. To study the mechanism of ARC-dependent recruitment of tankyrase binders and enable protein-observed NMR screening methods, we have as the first step obtained a full backbone and partial side chain assignment of TNKS2 ARC4. The assignment highlights some of the unusual structural features of the ARC domain.

Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase

“…To directly identify fragment binding sites on the ARC, we performed a full backbone and partial side-chain assignment of TNKS2 ARC4, doubly labelled with 15 N and 13 C isotopes. The assignment details have been reported elsewhere 61 . The control peptide induced significant chemical shift perturbations (CSPs), indicative of peptide binding in both a fast and slow kinetic regime (Supplementary Fig.…”

“…Doubly 15 N/ 13 C-labelled protein for the backbone and partial side-chain assignment of TNKS2 ARC4 was prepared as the 15 N-labelled protein, except that 13 C-D-glucose (Cambridge Isotope Laboratories; at 6 g/L of M9 media) was used as well. Method details have been reported elsewhere 61 .…”

Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase

“…We used the 3BP2 TBM peptide as a positive control. To directly identify fragment binding sites on the ARC, we performed a full backbone and partial side-chain assignment of TNKS2 ARC4, doubly labelled with 15 N and 13 C. The assignment details will be reported elsewhere (Zaleska et al, 2019) 69 . The spectra showed significant chemical shift perturbations (CSPs) induced by the peptide, indicative of peptide binding in both a fast and slow kinetic regime ( Supplementary Figure 3).…”

“…Overall, out of 165 amino acids (construct + 3 N-terminal residues introduced by the cloning method), 164 residues were assigned and backbone amides were missing for only two non-proline residues. Assignment details and methods will be reported elsewhere (Zaleska et al, 2019) 69 .…”

“…N-labeled protein, except that13 C-D-glucose (Cambridge Isotope Laboratories; at 6 g/L of M9 media) was used as well. Method details will be reported elsewhere(Zaleska et al, 2019) 69 .Kd determination using chemical shift perturbation TBM peptide and fragment titrations15 N-TNKS2 ARC4 (488-649) (300 µM final concentration; 5 µL of 3 mM stock) in NMR buffer (45 µL, 25 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 1 mM TCEP, 10% D2O) was used as the baseline sample. Peptide titration samples were prepared by diluting peptide (table 5) with NMR buffer (45 µL), then adding TNKS2 ARC4 (300 µM final concentration; 5 µL of 3 mM stock).…”