GAB2 is a scaffolding protein that, in the embryonic and postnatal development, is involved in various cellular processes, including cell proliferation, survival, migration, and differentiation. GAB2 is implicated in several cancers of both solid and haematological origin; being overexpressed in breast, gastric, lung and haematological cancers. This project is based on the hypothesis that an effective chemotherapeutic strategy would be that of interfering with the GAB2 protein-protein interaction network.
The structural architecture of GAB2 consists of an N-terminal PH domain (about 120 aa), while the reminder of the protein is essentially disordered (total length 676 amino acids). 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, and folds upon binding. Because of its highly dynamic properties, the structure of GAB2 has, to date, escaped an experimental characterization. The experimental plan is therefore to characterize by fluorescence monitored equilibrium and kinetics the interactions between GAB2 and its physiological partners and to pinpoint, by site-directed mutagenesis, the critical residues in the function of GAB2.
Activity 1: Site-directed mutagenesis to introduce fluorescent probes to study spectroscopically the interaction between GAB2 and its physiological SH2 and SH3 partners.
Because the project aims at understanding the details of the interaction between GAB2 and its physiological partners by studying the individual domains, it will be critical to be able to measure the reaction spectroscopically. We previously successfully tackled this problem with other IDP¿s by inserting a fluorescent residue in the proximity of the binding surfaces. We therefore plan to mutate the SH3 and SH2 domains of Grb2, SHP2, p85, PLC-g, CRK, SHC and SHIP and insert a tryptophan residue, which will allow to measure the binding reaction to GAB2. The tryptophan residues will be inserted at different alternative positions to be able to probe the folding upon binding reaction from different structural points.
Activity 2. Phi value analysis of the interaction between GAB2 and its physiological partners.
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 ¿ 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.
We will perform a complete phi value analysis of the interaction between the different regions of GAB2 (expressed as isolated peptides) and the SH3 and SH2 domains of Grb2, SHP2, p85, PLC-g, CRK, SHC and SHIP. We will produce a large number of point mutants and measure the effect on the kinetics and equilibrium of the reaction of folding upon binding. The effect of the mutations will allow to calculate the phi values and, therefore, to infer the presence and structure of any transient speicies to the highest experimental detail.
The kinetics of the reaction will be monitored by fluorescence monitored time resolved experiments. We will use both the stopped-flow (for the ms to s time range), and an in- house built continuous-flow and a temperature jump apparatus (for the sub ms time range). Equilibrium binding experiments will be performed both by measuring the fluorescence in titration experiments and by isothermal calorimetry. The parameters extracted from these experiments on all the mutants will be employed to calculate phi values, as previously described on other IDP systems (see for example Giri et al., 2013)
Activity 3 Determination of the structure of transition states and intermediates by restrained molecular dynamics simulations.
The structural information provided by phi values can be used to obtain structural ensembles representing the transition states for folding (Vendruscolo et al., 2001). In this approach, a trajectory is generated by integrating the equations of motion of a protein with a bias based on the incorporation of the phi values in the force field. This approach is analogous to the use of inter-atomic distances obtained through nuclear Overhauser effects (NOEs) to determine native state structures.
By using the results obtained in Activity 2, we will perform restrained molecular dynamics simulations and solve the structure of the reaction intermediates and transition states. This method will provide a complete structural depiction of the reaction pathways. These results will contribute i) to define the mechanism and energetics of interaction between GAB2 and its physiological partner, ii) to unveil the presence of any allosteric effect, iiii) to shed light on the role of any residual structure in GAB2 in tuning its physiological role, iv) to measure directly the strength of the contacts of each amino acidic position in the complexes between GAB2 and its partners.