Fig. 3: ALX4 cooperativity is facilitated by three protein-protein interfaces.

A, C, E Van der waals interactions were calculated via the total accessible surface area of the residue that is buried by the other protein chain and are shown in the bar graphs. Polar contacts are shown via dotted lines between interacting residues: hydrogen bond contacts are shown with black dotted lines, whereas salt bridge contacts are shown via red dotted lines. B, D, F Protein structure views of the protein-protein interfaces. A, B The N-terminal ARM (N-ARM) of Chain A contacts the turn between helix 1 and 2 as well as helix 3 of Chain B. C, D The start of both recognition helices forms a symmetrical interface. E, F The N-ARM of Chain B forms contacts with the turn between helix 1 and 2 of Chain A. G, H ALX4 protein (0, 50, 100, and 200 nM) was tested on the P3 fluorescent probe. Tau cooperativity factors were calculated for every lane in which protein was added. Bars depict the average Tau for each protein and each dot represents a Tau from an independent binding reaction. Error bars denote standard deviation. Tau factors (n = 6 binding reactions; biological replicates) were compared with a one-way ANOVA with a Holm-Bonferroni post-hoc correction. Note, quantitation was derived from two gels from the same experiment that were processed in parallel. G R215A (R2A), R216A (R3A), and R215A; R216A (R2A; R3A) mutations negatively affect ALX4 cooperativity. H D240G (D27G), V241R (V28R), and E255A (E42A) reduce ALX4 cooperativity. I ALX4 activates transcription in a cooperativity dependent manner. Luciferase assays were performed in transfected HEK293T cells. Bars denote average luciferase fold change of sample compared to reporter alone and dots represent an independent transfected well (n = 3 transfected wells; biological replicates). Error bars denote standard deviation and luciferase fold changes between proteins were compared via a one-way ANOVA with a Tukey post-hoc correction. A single replicate of each EMSA is shown and all replicates are in Supplementary Fig. 8. A–F Created in BioRender. Cain, B. (2025) https://BioRender.com/z65l674. Source Data are provided on Figshare⁶⁹.