Figure 2. EsxH_Mt binds Hrs.

(A) Gal4 DNA-binding domain (DB) fusions of EsxH_Mt, EsxH_Ms, or mutant EsxH_Mt were tested for Y2H interactions with Gal4 activation-domain (AD) fusions of EsxG_Mt, human (Hs), mouse (Mm), or zebrafish (Dr) Hrs. (B) Y2H interaction between indicated DB and AD constructs. Hrs is human. EsxG_Mt-EsxH_Mt-DB did not interact with EsxG_Mt-AD or EsxH_Mt-AD, presumably because of the intramolecular interaction in the DB construct. (C) Increasing amounts of Hrs were incubated with a constant amount of immobilized EsxG_Mt-EsxH_Mt and bound fraction examined by Coomassie blue. (D) Average binding (n = 3) was fitted with the Hill function, revealing a Hill coefficient of ∼1.7 and a K_D of 5.4 µM. (E) EsxG_Mt-EsxH_Mt-DB was tested in the Y2H for interactions with human Hrs-AD deletion constructs. The domain structure of Hrs is indicated.