Cell 147, 1340–1354 (2011)

Tankyrase (TNKS) is a poly(ADP-ribose)polymerase that transfers ADP-ribose onto protein substrates, regulating their stability and function. Although mutation of the TNKS-interaction site in the substrate 3BP2 underlies the human disease cherubism, little is known about the molecular determinants for substrate recognition. To address this gap, Guettler et al. evaluated the five substrate-binding ankyrin repeat clusters (ARCs) in TNKS2 to confirm at least four substrate binding sites. They solved the X-ray crystal structure of ARC4 with or without 3BP2 substrate peptide and used these structures to predict substrate-bound structures for the remaining ARCs. Four groups of substrate-contacting residues, including two regions that the authors termed an 'arginine cradle' and an 'aromatic glycine switch', were validated with mutagenesis and in multiple protein-protein interaction assays. X-ray crystal structures of ARC4 with peptides from additional protein substrates confirmed the generality of the configuration observed for ARC4-3BP2. Using a peptide library containing substrates that vary at positions in the recognition motif, the authors use fluorescence polarization assays to confirm a requirement for arginine and glycine residues at positions 1 and 6, respectively, and use a position-specific scoring matrix to predict new substrates. Coimmunoprecipitations confirmed 13 new TNKS2 interactors as well as the dependence of these protein-protein interactions on the recognition motif. Together, these data provide the first molecular-level view of tankyrase-substrate interaction and predict additional substrates for these enzymes.