Figure 3. Interrogation of TNKS2^ARC4-binding peptides by SIMBA.

(A) Heatmap showing effects of all single-position substitutions in the 3BP2 motif. Colors denote enrichment (red) or depletion (blue) relative to the wild-type motif, calculated as log scores by Enrich2 software (see Methods). The asterisk denotes a termination codon. The data average four independent experiments using P_GAL1-driven TNKS2^ARC4-Cln2 (two each of N-site and C-site libraries).

(B) Logos showing TNKS2^ARC4 sequence preferences observed for the 3BP2 motif in vitro [54] and in vivo (this study), plus the average in vivo results from 5 distinct motifs (see also Figure S2B–D). For clarity, only positive preferences are shown (see Methods).

(C) Plots of SIMBA scores vs. K_D for all 3BP2 variants as well as representative individual positions. Yellow, wild-type; pink, missense variants. SIMBA scores are mean ± SEM (n = 4). K_D values [54] are the mean of technical duplicates (n = 1 ± SE of the regression fit); K_D values that were not quantifiable in vitro were assigned values of 200 μM to allow inclusion in the plot. See also Figure S2A.

(D) SIMBA scores vs. K_D values [54] for the wild-type AXIN1 peptide plus 9 Ala mutants, plotted as in panel (C). See also Figure S2B–D.

(E) Comparison of STOP-normalized SIMBA scores to in vitro K_D [54] for 6 wild-type peptides. See also Figure S2B–D.

(F) Diagram of N-site and C-site locations for inserting SLiM sequences.

(G) Correlation of SIMBA scores for variants of the RPGopt motif in N-site vs. C-site locations. Data are the mean ± range (n = 2). See also Figure S2E.

(H) Correlation of SIMBA scores for RPGopt motif variants when the TNKS2^ARC4-Cln2 fusion was expressed from different strength promoters (P_GAL1 vs. P_GALS). Data are the mean ± SEM (n = 4; two each of N-site and C-site libraries). See also Figure S2F.

(I) Summary of Pearson correlation coefficients (r) for the indicated pairwise comparisons. See also Figures S2E–F.