Fig. 6. The thioredoxin-like fold of SasA and fsKaiB cooperatively recruits KaiB to the KaiC hexamer.

(A and B) Equilibrium binding assays of fluorescently labeled KaiB with KaiC-EE in the absence (black) or presence of (A) unlabeled SasA (light to dark purple) or (B) fsKaiB (light to dark orange). Each 2D titration is shown from representative assays with best fit lines from global curve-fitting. (C) Summary of a two-site thermodynamic model for SasA heterocooperativity, and mean equilibrium constants derived from least-squares fitting of SasA 2D titration assays (n = 3); values in red are mean peak anisotropy values observed from fitting of all SasA 2D titration datasets using DynaFit software. The cooperativity index is defined as the ratio K₁/K₃ = K₂/K₄. This model was used to generate simulated 2D titrations with a cooperativity index of either 10 (D) or 1 (E). (See data S6 to S8 for DynaFit scripts and full datasets.)