(A) Affinity of CID/NCBD complexes was measured by isothermal titration calorimetry (three examples are shown including the low-affinity D/P NCBD, 1R CID interaction). (B) The affinities (Kd values) were normalized against the interaction between extant human NCOA2 CID and p300 NCBD. The relative affinity for D/P NCBD, 1R CID was calculated using the average Kd values of all D/P NCBD variants (5 ± 2 µM). (C) Propensity for helix formation for ancient and extant CID domains as measured by circular dichroism at 222 nm upon addition of the helix stabilizer 1,1,1-trifluoroethanol. (D) Global stability of NCBD domains as measured by circular dichroism at 222 nm (reflecting the fraction folded NCBD) upon addition of the denaturant urea. Hsa, Homo sapiens; Dre, Danio rerio (zebrafish); Pme, Petromyzon marinus, (sea lamprey); Dmel, Drosophila melanogaster (fruit fly).