Fig. 3: Interaction between the CH and the bMERB domain of EHBP1 and disruption by Rab8.

a The bMERB domain (green), CH domain (orange), and a mixture of both (blue) were loaded onto a Superdex 75 10/300 GL column to monitor for complex formation. Clear complex formation was observed for the construct lacking the C-terminal helix (indicated as green helices in the insets). However, in the case of the full-length construct, only a shift in the CH domain peak was observed, indicating distribution between free and EHBP1 bMERB bound CH domain at the concentrations used. No complex formation or shift in the CH peak was observed for the construct lacking the N-terminal helix. b Binding affinities were measured by titrating the CH domain (800 µM) to either the full length or N/C-terminally truncated bMERB domain (60 µM). Integrated heat peaks were fitted to a one-site-binding model yielding the binding stoichiometry (N), the enthalpy (ΔH), the entropy (ΔS), and the dissociation constant (K_D). The data are representative of at least three repetitions. c Observed association first-order rate constants between 0.5 μM Cy3 labeled CH domain with different concentrations of full-length bMERB domain (2–16 µM). Kinetics were registered as a change in fluorescence using a stopped-flow apparatus at 25 °C. Association of bMERB to the Cy3-CH leads to a decrease in the fluorescence. As an example, the kinetics of association between 0.5 μM Cy3 labeled CH domain and 5 μM of bMERB domain is shown in the inset. d, e Dissociation of the CH domain was measured by monitoring the increase of fluorescence after mixing a complex of Cy3 labeled CH with full-length bMERB domain (2 μM) with a 20-fold excess of either unlabeled CH domain or GppNHp Rab8a. f Results of systematic analysis of interactions between the bMERB domains of different family members with their respective CH/LIM/CH-LIM domains (from Supplementary Fig. 2). Interactions were measured by ITC. N.D. denotes not detected.