Abstract
Gab2 is a large disordered protein that regulates several cellular signalling pathways and is overexpressed in different forms of cancer. Because of its disordered nature, a detailed characterization of the mechanisms of recognition between Gab2 and its physiological partners is particularly difficult. Here we provide a detailed kinetic characterization of the binding reaction between Gab2 and the C-terminal SH3 domain of the growth factor receptor-bound protein 2 (Grb2). We demonstrate that Gab2 folds upon binding following an induced fit type mechanism, whereby recognition is characterized by the formation of an intermediate, in which Gab2 is primarily disordered. In this scenario, folding of Gab2 into the bound conformation occurs only after binding. However, an alanine scanning of the proline residues of Gab2 suggests that the intermediate contains some degree of native-like structure, which might play a role for the recognition event to take place. The results, which represent a fundamental step forward in the understanding of this functional protein-protein interaction, are discussed on the light of previous structural works on these proteins.
Highlights
The growth factor receptor-bound protein 2 (Grb2)-associated binding protein 2 (Gab2) is 74 kDa scaffolding protein that is involved in functional cellular signalling and cancer development [1,2,3,4]
We show that a comparison between the experiments performed by varying the concentration of Gab2503-524 or Grb2 SH3C reveals unambiguously that the reaction occurs via an induced fit type mechanism, whereby the folding of Gab2503-524 to a polyproline type II structure is subsequent to the formation of an initial encounter complex, despite its partial character of polyproline II when in the unbound
In an effort to understand the order of the events in the folding reaction of Gab2503-524 upon binding to Grb2 SH3C, we resorted to apply a kinetic test, originally introduced by Olson et al [31] and more recently applied on different protein systems [32,33,34]
Summary
The Grb2-associated binding protein 2 (Gab2) is 74 kDa scaffolding protein that is involved in functional cellular signalling and cancer development [1,2,3,4]. Whilst the functions of Gab are critical in the embryonic and postnatal development, its expression in a healthy mature cell is relatively suppressed. For these reasons, targeting Gab provides a promising route to develop new strategies for highly selective drug therapies in anticancer treatment. This protein acts at the head of signalling networks, by conveying inputs from membrane receptors to the cytoplasm, where it dictates the functions of multiple pathways
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