Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Understanding the folding and binding mechanisms of SH2 domains

The interaction between proteins plays a paramount role in biological functions. Frequently, protein-protein interactions are mediated by specific classes of domains that can recognize a determined consensus sequence. To avoid the misregulation of cell physiology, different protein-protein interaction domains evolved to ensure specificity and proper affinity for their natural ligand(s). Since many signalling pathways are mediated by tyrosine phosphorylation, their regulation is controlled in many cases by specific domains able to recognize phosphorylated tyrosine, the so-called SH2 domains. Proteins that contain such domains can minimize cross-reactivity with non-desired targets thanks to the high specificity of the SH2. The understanding of many complex biological processes relies on our ability to describe the underlying fundamental processes. In this context, the study on the stability and functions of isolated domain plays a critical role in modern molecular biology and many signalling pathways have been successfully described by defining the interaction networks of individual domain. For this reason the aim of this thesis is focused on the description of the mechanisms by which proteins of the SH2 family recognize their specific partners in normal and pathological conditions. Furthermore, we addressed the effect of site directed mutagenesis on the folding and stability of this important class of protein domains. Through a combination of site-directed mutagenesis and biophysical techniques such as Stopped-flow kinetics, and NMR, we characterized the mechanism of folding and binding of different SH2 domains, highlighting some possible general characteristics for this class of proteins. In spite of their importance for cell physiology, there is a lack of information pertaining the details of the mechanism of binding of SH2 domains. Our results highlighted for the first time an allosteric mechanism of interaction for the SH2, which we speculate may represent a possible general mechanism of interaction for this class of proteins. Such allosteric mechanism is characterized by a remarkable structural plasticity, with the binding reaction being mediated by a diffused structural region and not solely by the residues located in the binding pocket. In addition through a biophysical approach named double mutant cycle, we defined potential allosteric sites in the SH2 domain from PI3K, which mediate and control the interaction with the partner. Future investigations will be used to explore more in details the allosteric nature of the binding process of other SH2 domains, with the final aim of defining allosteric sites, which could potentially be used as therapeutic targets. The extensive studied on SH2 domains allowed us to also define the molecular mechanism of a specific mutant of the N-terminal SH2 domain from the protein SHP2 (the T42A mutant), which has been demonstrated to be at the basis of the onset of the Noonan Syndrome. Analysing the binding properties of N-SH2 T42A we defined the molecular details that could explain the role of N-SH2 T42 in the disease and so we provided a possible explanation for the hyper-activation of SHP2 in the Noonan Syndrome.