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Whereas the peptide corresponding to 3BP2 bound with an affinity of 4.9 �� 0.4?��M, the sequence-optimized peptide bound almost an order more tightly, with an affinity of 0.6 �� 0.04?��M (Figure?5B). Deviations from the superconsensus at any position, individually or jointly, may allow the affinity of substrate-targeting peptides to be differentially tuned, as appears to be the case. Isoleucine at position 5 conferred weak binding (KD?= 72.4 �� Selleck VX 770 31.6?��M) in the screen, but the TNKS-binding motif peptide of MCL1, which contains isoleucine at position 5, bound the ARC robustly (KD?= 2.4 �� 0.2?��M). Similarly, valine at position 4 or proline at position 5 were disallowed/unfavorable in our screen, but AXIN1, another robust ARC binder (KD?= 6.1 �� 0.5?��M), contains exactly these residues at positions 4 and 5. Both of these TNKS-binding motifs contain the stabilizing glutamate residue at position 8, which can form a salt bridge interaction with K604TNKS2 that likely offsets the negative impact of suboptimal amino acids at positions 4 and 5. The targeting peptides of both MCL1 and AXIN1 are indeed highly sensitive to the K604A mutation in ARC4 (Figure?S4C and Table S2). Furthermore, although introduction of the AXIN-like residues valine and proline at positions 4 and 5, respectively, Protein Tyrosine Kinase inhibitor into the TNKS-binding motif of 3BP2 abolished measurable binding, binding was partially restored when either glutamate or aspartate, but not glutamine or asparagine, was present at position 8 (Figure?S6C). To identify candidate Tankyrase binders, we performed an in?silico search of the human proteome, using a position-specific scoring matrix (PSSM) approach based on the affinity data obtained Ribonucleotide reductase in the peptide library screen (Yaffe et?al., 2001; see Experimental Procedures for details). We calculated a Tankyrase-targeting score (TTS) of minimally ?0.817 (no match) to maximally 1 (best match) for each peptide in the UniProt database (octapeptide scan with additional scoring of C-terminal hepta- and hexapeptides; see http://research.lunenfeld.ca/sicheri/files/file/PSSM_raw_list.txt.zip for a list of all peptides scored). The vast majority (10,420,456; 93.7%) of the 11,124,194 peptides scanned obtained a low TTS of