TITLE: Predicting Tankyrase Binders
AUTHORS: Katie Pollock, Michael Ranes, Ian Collins, Sebastian Guettler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is part of a collection: </span><a href="https://www.protocols.io/private/DA5F6F33163011EBAE360A58A9FEAC2A" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach</span></a></div><br/><div class = "text-block"><span>The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from </span><span style = "font-style:italic;">Escherichia coli </span><span>and outline a fluorescence polarization assay to quantitatively assess direct ARC–TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells.</span></div></div>

[STEPS]
?. [Predicting Tankyrase Binders]
Screen your protein of interest for potential TBMs. In an initial step, a search for Arg and Gly spaced 6 amino acids apart (i.e., with four amino acids in between, RxxxxG) will suffice. See step 4 below for atypical TBMs.
?. [Predicting Tankyrase Binders]
Using UniProt (http://www.uniprot.org), assess the topology of the protein and whether the candidate motif would be located in the cytoplasm or nucleoplasm, accessible to tankyrase [24]. The relevant information can be found under Subcellular location > Topology.
?. [Predicting Tankyrase Binders]
Assess the occurrence of disordered stretches in your protein of interest. TBMs must be present in unstructured regions of the protein to be accessible to tankyrase and must not be part of a stable α-helix or β-strand. Secondary structure information, obtained from the Protein Data Bank [25], is available in UniProt (http://www.uniprot.org) under Structure > Secondary structure [24]. It remains possible that TBMs are present within loops of structured protein domains. If no structural information is available, a disorder predictor such as IUPred (http://iupred.enzim.hu) may be used to obtain an indication of protein disorder [26].
?. [Predicting Tankyrase Binders]
Assess the short-listed sequences for their suitability as TBMs. REAGDGEE has been found to be the optimal 8-amino-acid TBM by positional scanning [7], but many TBMs only span six amino acids (positions 1–6). While the sequence rules from positional scanning prove valuable in predicting TBMs, it should be noted that some of their aspects may be specific to the context of the 3BP2 TBM peptide , which positional scanning was performed on [7]. Whereas Arg and Gly at positions 1 and 6, respectively, are essential, other residues are important as well, with the exception of positions 2 and 3, which can likely be ignored since they do not contribute to binding and mainly fulfil spacing roles (Fig.1b, d). Please refer to the motif description under Subheading 1 (Introduction) for the assessment. Of note, a large or strongly charged amino acid at position 4 very likely disqualifies the TBM candidate as a genuine tankyrase binder. Furthermore, Asp and Glu at position 8 can compensate for unfavorable amino acids at other positions [7], as seen in one of the TBMs of AXIN1/2, where a suboptimal Val at position 4 appears to be counterbalanced by a Glu at position 8 (Fig.1e) [7, 11]. Interestingly, a second TBM in AXIN1/2 shows that the Arg, normally at position 1, can be placed further N-terminal, resulting in looping-out of the “inserted” residues (Fig.1c, e). Likewise, the E3 ubiquitin ligase RNF146 has been proposed to contain numerous atypical TBMs with a nonconventional positioning of the Arg [27], and it is possible that a distal Arg is required for weak binding of the TBM of GRB14 to tankyrase (Fig.1e) [7, 8, 28]. Thus, the possibility of a distal Arg residue should be taken into consideration. It is likely that such extended TBMs display a weaker affinity for tankyrase. Numerous tankyrase binders contain multiple TBMs, such as AXIN1/2 [11] and MERIT40 [7] (see below). Since avidity effects likely contribute to binding of these proteins to tankyrase, the affinity of some of these individual TBMs for the ARC may be lower, and this may be reflected in deviations from the “optimal” sequence.