CRKL, GAB2, and FRS2 are scaffolding adaptor proteins that are involved in various cellular processes, including cell proliferation, survival, migration, and differentiation. They are implicated in several malignancies, being recurrently amplified in several types of cancers and essential to cancer cell lines that harbor such amplification.
The function of the adaptor proteins CRKL, GAB2 and FRS2 is primarily mediated by their capability to assemble different proteins in specific complexes. This feature is provided by their modular structure containing SH2, SH3 and/or PTB domains, as well as proline-rich sequences (targeted by SH3 domains) or phospho-tyrosines (targeted by SH2 or PTB domains). Of additional interest, despite large portions of CRKL, GAB2 and FRS2 are disordered, these parts are nevertheless functional and extremely important as they mediate the interaction with their partners. Because of its highly dynamic properties, the structure of CRKL, GAB2 and FRS2 has, to date, escaped an experimental characterization. The project is therefore aimed at:
i) determining the structural features of the disordered regions of CRKL, GAB2 and FRS2 implicated in binding
ii) studying the structure of the complexes between the relevant domains of the physiological partners and the interacting regions of CRKL, GAB2 and FRS2
iii) characterizing the interactions between CRKL, GAB2 and FRS2 and their physiological partners
iv) pinpointing, by site-directed mutagenesis, the critical residues in the function of CRKL, GAB2 and FRS2
v) exploit structural and mechanistic knowledge to derive an in silico pharmacophore model, in order to find potential inhibitors of protein-protein interactions to be tested both in vitro and in cellula
As detailed in the Plan, uncoupling CRKL, GAB2 and FRS2 would result into the equivalent of having a cocktail of drugs targeting multiple enzymes, because their activation could be prevented simultaneously.
The classical view on proteins is deeply affected by the belief that a well-defined three dimensional structure is a pre-requisite for their function. Consequently, the past ten decades have witnessed a continuous advance of structural biology, with the aim of depicting the structure of very complex protein architectures. Notably, one of the most important finding in recent biology has not been represented by the description of a beautiful fold, but rather by the observation that many proteins, or portions of them, lack a well-defined structure and appear highly dynamic. While disordered, these proteins may mediate many critical cellular functions, such as intracellular signaling, transcription and replication. Often, these intrinsically unstructured systems fold upon binding to their target ligand, thereby undergoing a binding induced folding reaction. The concept of functional disorder is slowly percolating in the scientific community and many untested hypotheses have been put forward to explain its role in the living systems. The most relevant theories are: i) It is a way of decoupling affinity and specificity; ii) It increases the association rate with the ligand; iii) It allows for increased plasticity with regard to the ligand; iv) A large interaction surface area is provided in a short amino acid sequence as the protein folds around its ligand; v) Rapid turnover in the cell. Despite all these hypotheses are intriguing and would corroborate a potential value for protein disorder, there is a paucity of experimental data on the mechanisms by which intrinsically denatured systems recognize their partners.
The submitted Research Project represents an opportunity to infer the properties of intrisincally disordered proteins, in the context of a scientific problem intimately linked with disease. A major ground of innovation lies therefore in providing the molecular understanding on the function of protein disorder, while tackling important protein systems linked with disease.
The project will tackle for the first time the mechanistic details of the mechanisms of interactions between disordered segments of the adaptor proteins and their binding partners. These aims will represent a relevant step forward in the understanding the role of these systems in cancer. In fact, scarse biophysical data is available to date on the molecular basis of these interactions.
Furthermore, the computational selection and in vitro testing of possible drugs to modulate the function of CRKL, GAB2 and FRS2 represents a tantalizing possibility to relieve cancer. Whilst such a goal is relatively ambitious, appears rather feasible as the experimantal approach described in the Project has been previously successful on similar small domain protein systems.