Fig. 2: ALX4 cooperativity is DNA mediated.
From: The ALX4 dimer structure provides insight into how disease alleles impact function
A A map of the ALX4 HD with the ALX4 amino acid numbering (ALX4 #) and canonical homeodomain numbering (HD #) below. Created in BioRender. Cain, B. (2025) https://BioRender.com/t12p127. B The ALX4 HD is sufficient to mediate cooperativity on the P3 site but is unable to bind cooperatively to the P4 site. ALX4 protein (0, 37.5, 75, 150, and 300 nM) was combined with each respective fluorescent probe. A single replicate is shown here, and all three replicates are shown in Supplementary Fig. 6A. Schematics of the protein-DNA complexes are shown to the right of the gel. C Tau cooperativity factors were calculated for every lane in which protein was added. Bars depict the average Tau for each probe and each dot represents a Tau from an independent binding reaction (n = 12 binding reactions; biological replicates). Error bars denote standard deviation. Tau factors were compared with an unpaired, two-sided student t-test. Note, quantitation was derived from two gels from the same experiment that were processed in parallel. D X-ray structure of ALX4 reveals that ALX4 binds as a dimer to the P3 site in a head-to-head orientation. ALX4 Chains A and B are colored cyan and green, respectively; DNA is colored gray. ALX4 residues R218 (canonical HD numbering: R5), Q263 (Q50), and N264 (N51) are shown in a stick representation. E Schematic of ALX4-DNA interactions. Hydrogen bond interactions were determined by PDBePISA and required atoms to be within 4 Å. Chain A and B proteins are labeled in blue and green respectively. Hydrogen bond interactions to the DNA backbone are shown via black dotted lines whereas hydrogen bonds to nucleotide bases are shown in red. Created in BioRender. Cain, B. (2025) https://BioRender.com/a69r574. F, G Structural comparisons of protein-DNA interactions made by the ALX4 Chains A and B. Source Data are provided on Figshare69.
