Fig. 3. The HOX domain drives stable association of OC2 to DNA.

a–c ITC binding analysis of intact OC2, CUT domain and CUT + HOX, to PEG10 DNA. The raw heats (differential power, DP; top) and binding isotherms (bottom) are shown and representative of three independent experiments (n = 3; technical replicates). Source data are provided as a Source Data file. d–g Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) characterization of DNA binding by OC2. Structure of OC2 bound to PEG10 DNA showing the helix α3 (purple) which appears to become much more structured upon PEG10 binding (d). The CUT and HOX are in green and gold, respectively, while the PEG10 is in wheat. The protein N- and C-termini are indicated. Relative deuterium uptake into OC2 with and without bound PEG10 is rendered on the structure using a blue-white-red gradient scale (e); regions of the protein not covered by the HDX-MS experiment are in black. No regions of increased uptake were observed but many parts of the protein experienced decreased (blue) deuterium uptake upon PEG10 binding with the maximum decrease being 0.28. Relative deuterium uptake into the CUT domain alone with and without bound PEG10 is plotted on the structure according to the same scale and color scheme as in panel e (f). There were no significant differences in deuterium uptake upon PEG10 binding of the CUT domain alone (consequently, mostly white except the black regions that showed no coverage). Note that the CUT(-DNA) structure is extracted from the OC2-PEG10 structure for demonstration only. Deuterium uptake plot for the helix α3 containing peptide (residues 358-371) in the four different conditions as indicated (g). The y-axis corresponds to the total number of amides in the peptide. The HDX-MS data shown is representative of three independent experiments (n = 3; technical replicates); P-values based on One-way ANOVA as provided by the software were 0.001 for OC2-PEG10 vs apo-OC2; 0.9 for CUT-PEG10 vs apo-CUT.