Fig 2: Tandem ALDLS motif-containing RAI2 binding to CtBP is autonomous.a Protein constructs in proportion to their length. Colors: RAI2, yellow: ALDLS motifs 1 and 2, orange; CtBP1 and CtBP2: split PxDLS binding domain (BD), blue; dehydrogenase domain (DD), pink; C-terminal domain (CTD), gray; nuclear localization sequence (NLS), red. CTD gray shades of CtBP1 and CtBP2 indicate CTD diversity. b CD spectra of CtBP1(WT, FL) (blue), RAI2(WT, 303-465) (orange) and CtBP1(WT, FL)/RAI2(WT, 303-465) complex (green). c SAXS Kratky plots of the protein constructs used in (b) indicating the level of structural flexibility and fold content. The plots are based on a single experiment each and the error bars represent the propagated SEM of the scattering intensities measured at each point following Poisson counting statistics90. The data have been taken from SASBDB entries SASDQW5, SASDQ46, and SASDQC6. d Dissociation constants KD (left panel) and stoichiometry values (right panel) of RAI2(303–362) WT, M1 and M2 to CtBP1. The data are represented as mean of n = 3 independent biological replicates ± SD. Unpaired t-test was used to evaluate statistical significance applying p values for a two-sided confidence interval as follows: p < 0.001 (***), p < 0.01 (**), p < 0.05 (*). Definition p values: KD: p = 0.0017 M1, p = 0.0007; Stoichiometry: p = 0.0001 M1, p = 0.0217 M2. e Sequence of RAI2(WT, 303–362, yellow) comprising the twofold repeated ALDLS motif (orange). Residues mutated in RAI2 M1, M2 and M1 + M2 variants are in red. Removed linker residues in RAI2(Δ11, 303–362) are in light yellow and are indicated by a double arrow. f HSQC spectra of RAI2(WT, 303–362). g Effects of CtBP1 titration on selected RAI2 residues (boxed and labeled in F) in M1 (left) and M2 (right) variants. Colors of increasing amounts of titrated CtBP1 (WT, 28–353) on RAI2(303–362) variants: orange (1:0), dark gray (1:0.4), light gray (1:0.7), and green (1:1). h Histogram of chemical shift perturbation intensity ratios upon titration of CtBP1(28–353) onto RAI2(303–362) variants (WT, M1, M2). Mutated ALDLS sites are in gray. Low intensity ratios indicate perturbation of RAI2 residues in the presence of CtBP. See also Supplementary Figs. 1–5, Supplementary Table 1 and Supplementary Data 1, 3–5, and 8. Source data are provided as a Source Data file.

Fig 3: Mechanisms model for the regulation of RAI2 in Wnt/β-catenin signaling. ① RAI2 inhibits the interaction of CtBP2 and APC. ② RAI2 suppresses the expression of CtBP2.

Fig 4: Annotation of KEGG pathway among the top 30 samples with the highest and lowest RAI2 expression via multiple GSEA analysis. (A) Pathways positively correlated with RAI2 expression. (B) Pathways negatively correlated with RAI2 expression.

Fig 5: Molecular basis of RAI2-induced CtBP polymerization.a Cryo-EM density (left), schematic (middle), and cartoon representation of the CtBP1(WT, FL)/RAI2(Δ11, 303–362) filament confined to six CtBP1 tetramer layers (right) that are colored in cyan, violet, and light blue. Adjacent CtBP1 layers are rotated around a vertical 3-fold filament axis and are connected by a pair of RAI2 peptides (yellow) with an ALDLS tandem motif (orange) in opposite positions to the central filament axis. Book-like opening of a CtBP1(n)/CtBP1(n + 1) interface (boxed) without bound RAI2 is indicated schematically. b Top view of the RAI2-mediated CtBP1(n)/CtBP1(n + 1) interface in cyan and pale cyan (boxed in a, left). Areas interacting with the next tetrameric CtBP layer are in blue and dark blue. The four CtBP subunits are numbered as in Fig. 2e. Residues involved in specific CtBP1/CtBP1 interface interactions are indicated (cf. c). RAI2-interacting surface segments are in orange (ALDLS motif) and yellow (other residue segments). c Side view of the CtBP1(n)/CtBP1(n + 1) interface (boxed in a, left), highlighting secondary structural elements and residues that contribute to specific interface interactions (cf. Supplementary Fig. 13). d Side view of the visible RAI2 segment interacting with two adjacent CtBP layers (boxed in a, right). The connecting RAI2 tandem ALDLS linker (yellow) is in close contact with a third CtBP subunit (violet). See also Supplementary Figs. 10–14 and Supplementary Tables 3 and 4.