STRUCTURAL AND FUNCTIONAL STUDIES ON PROTEINS RELATED TO HUMAN DISEASES

Protein-protein interactions (PPIs) represent a vast and complex network of intermolecular relationships essential for organism, in which they control a wide range of biological processes. This network plays a vital role in the initiation and progression of cancer; hence, its understanding is crucial for identifying the key functional modulators of tumor progression and metastasis, and for the therapeutic intervention. During my PhD, I had the opportunity to contribute to the structural and functional studies on two different PPIs related to human cancer, the human thymidylate synthase (hTS) homodimer and the heterodimeric interaction between human yes-associated protein (hYAP) and human transcriptional enhanced associate domain 4 (hTEAD4). In the first chapter, the monomer-monomer interface of hTS was exploited to determine and deeply investigate residues critical for the quaternary assembly of the functional enzyme, employing a mutagenic approach. Hence, two sets of hTS interface variants, defined alanine and charged interface variants, have been generated and investigated through circular dichroism (CD) thermal denaturation, kinetic analyses, and X-ray crystallography. We have studied the two interface hot spots F59 and Y202, for which previous studies demonstrated the importance in the hTS dimerization process. The hTS variants F59A and Y202A have been analyzed by means of their thermal stability profiles in comparison to the native enzyme, and by attempting/performing their structural characterization. Notably, the crystallographic structure of the Y202A mutant has been also determined in complex with a dimer disrupter inhibitor, here named compound 1, providing significant information for the rational design of novel hTS interface-targeting molecules. In the second approach, we pointed the attention on the interface residues Gln62 and Thr251 facing themselves on the dimer halves, which have been mutated into Arg and Glu, respectively. The introduction of these charged amino acidic residues affects both the enzyme activity and stability. Our mutagenic approach successfully led to the destabilization of the hTS homodimer, as shown by their structural analyses, evidencing a slight aperture of their quaternary assembly,localized in the interface areas surrounding the mutated residues, also perturbing the active/inactive conformational equilibrium of the enzyme. The interactions of the new variants with the physiological substrate dUMP and its analogue FdUMP have been further characterized. Thus, our hTS interface variants, having a more accessible monomer-monomer interface area may represent useful tools exploitable to identify and screen innovative interface-targeting inhibitors. In the second chapter, the heterodimeric interaction between the Hippo-pathway terminal effector proteins, hYAP and hTEAD4, was examined. To expand the limited knowledges on this key PPI for tumorigenesis and on its modulation, we developed protocols for co-expression, co-purification and crystallization of hYAP TEAD-binding domain (TBD): hTEAD4 YAP-binding domain (YBD) complexes (named S, M, and L complexes). By means of X-ray crystallography, the structure of complex S has been solved, defining new targetable interfaces between the partner proteins. Notably, this work reports the development of the first protocol for the heterologous co-expression and co-purification of full-length hYAP in complex with hTEAD4-YBD (named XL complex), as well as its preliminary structural characterizations. The pilot data here obtained on the hYAP:hTEAD4 complex XL proposes the formation of extended interface areas, larger than those occurring in the complex with the hYAP TBD fragment. The study of these complexes provides interesting new clues helpful for the rational design and the development of molecules able to modulate this PPI playing a critical role in wide-range forms of cancer