Gab2, a key member of the Gab family of proteins, is a scaffolding protein involved in the amplification and integration of signal transduction. For this reason Gab2 is implicated in several cellular functions such as cell proliferation, survival, migration and differentiation. Gab 2 has been associated to several cancers of both solid and haematological origin. In particular Gab2 is overexpressed in breast, gastric, and lung cancers.
From a structural point of view, Gab2 is composed by 676 amino acids organized in a folded N-terminal PH domain (of about 120 amino acids) and a disordered C-terminal region. Despite this large portion of Gab2 is disordered, it is nevertheless functional and extremely important, as it mediates directly the interaction with the many cytoplasmic partners, containing SH2 or SH3 domains. Upon activation of some receptor tyrosine kinases (RTKs), the scaffold Gab2 becomes tyrosyl phosphorylated and interacts with SH2 domain containing signal relay molecules, including the p85 subunit of phosphoinositide-3-kinase (PI3K).
PI3K protein is an important physiological partner of Gab2 and it is composed by a regulatory subunit called p85 and a catalytic subunit called p110, that can catalyze the formation of the lipid membrane PIP3. Gab2 can directly interact with PIP3, remaining anchored to the membrane and allowing the correct transduction of the signal.
This project is based on the hypothesis that an effective chemotherapeutic strategy would be that of interfering with the Gab2/PI3K interaction. Hence, we plan to address the mechanism of interaction between the disordered regions of Gab2 and the SH2 domains of PI3K, with the final goal of designing specific inhibitors to hijack such interactions.
The detailed understanding of the binding mechanism between two proteins, requires the definition of the molecular bases that drive the recognition between the two proteins. Proteins that are able to interact with different partners, usually control their activity through conformational changes generally induced by covalent modifications or by the binding with allosteric effectors. However, in the literature, some examples are reported that show how the cis/trans isomerization of a single peptidyl-prolyl imide bond, can represent a mechanism by which proteins regulate their activity (Mallis R. J. et al. 2002; Lummis S. C. R. et al. 2005; Sarkar P. et al. 2007; Severin A. et al. 2009). In the work of Mallis et al., for example, it has been observed that the cis/trans isomerization of a single peptidyl-prolyl imide bond of the SH2 domain of the Itk protein, drastically affects the function of this domain. More precisely, they have demonstrated how this SH2 domain interacts with different parnters according to whether the peptidyl-prolyl imide bond is in a cis or in a trans conformation.
From preliminary studies conducted analyzing the structures of the two SH2 domains of PI3K, we observed the presence of a cis peptidyl-prolyl imide bond conserved in both domains. For this reason, one of our goal is to understand if the eventual isomerization of such bonds can represent a mechanism of control with which the PI3K protein regulates the binding with Gab2. Such evidence could be very important both for the design of a drug that prevents this interaction, and in further defining a mechanism of control of the protein function still poorly characterized.
The others principal goals of this project will be:
i) address the binding mechanism of isolated SH2 domains from
PI3K by using peptide mimicking their specific binding site in Gab2. This goal will be achieved by fluorescence monitored equilibrium and kinetics experiments.
ii) infer, by site directed mutagenesis, the interactions critical for the molecular recognition between Gab2 and its different partners.
iii)Phi value analysis of the interaction between Gab2 and the two SH2 domains of PI3K.
One of the most ambitious goals, when addressing the mechanism of a given reaction, is to provide a structural characterization of all the intermediates involved as well as the intervening transition state(s). To achieve this, we plan to use a powerful method, known as the phi value analysis (briefly discussed below), which we used successfully in the past on different protein systems (Gianni et al., 2015).
The method is based on the simple assumption that, by systematically mutating protein residues, while probing the effect of the mutation on the folding kinetics and ground state stability, it is possible to map, one by one, interaction patterns in the transition states. In fact, mutations that destabilize the transition state (or an intermediate) target contacts that are formed in its structure. The relative formation of the contact is commonly called the phi value. By producing and characterizing a large number of point mutants in a given protein it is therefore possible to draw a structural map of the transition and intermediate state(s) of a reaction, with detection of native like (phi values tending to 1) and denatured like (phi values tending to 0) clusters.
iv) Structural characterization of the complexes between Gab2 and the two SH2 domains of PI3K by NMR.
v) design efficient inhibitors aimed at either maintaining Gab2 in its unstructured, and unbound, state or at interfering with their binding mechanisms with the SH2 domains. The inhibitors, typically peptides, will be designed based on the experimental knowledge obtained from points i-iv.