A Dock and Coalesce Mechanism Driven by Hydrophobic Interactions Governs Cdc42 Binding with its Effector Protein ACK

Tetley GJN, et al., doi: 10.1074/jbc.M117.789883, J Biol Chem, 2017

Cdc42 is a s small G protein belonging to the Rho-family which has been widely studied for its role in controlling the actin cytoskeleton and playing an taking part in several potentially oncogenic signaling networks. The aim of this study was to complete description of Cdc42-binding site on ACK. Data suggested that the binding affinity of ACK relies on several conserved residues that are critical for stabilizing the quaternary structure. ForteBio’s Octet Red system was applied to study interactions between Cdc42 and GST fusion constructs of ACK, WASP, and a basic region mutant of WASP. GST sensors were used for immobilization of GST-tagged proteins. The analysis was performed at 25 °C in the following buffer: 50mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.02% Tween 20, 0.1% BSA, and 0.05% sodium azide. Data were collected for sensors loaded with GST alone and this was subtracted from the experimental traces (by using double referencing) to subtract the effects of nonspecific binding. Sensors were regenerated in 10 mM glycine, pH 2. Interaction analysis was conducted using BLI and SPA (direct scintillation proximity assay) method and results on both platforms were fairly comparable.

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