Dottorato in BIOCHIMICA

Titolo della tesi: Unravelling the mechanistic details of a protein recognition domain interactions with short linear motifs: binding and inhibition mechanisms

Protein-protein interactions are crucial in various biological processes, including metabolic pathways, cell cycle progression, signal transduction, and proteasomal systems. For PPIs to fulfill their biological functions, it requires the specific recognition of many interacting partners. In many cases, however, protein-protein interaction domains can bind different partners in the intracellular environment, demanding a fine regulation of the binding events to exert their functions correctly and avoid misregulating critical molecular pathways. In this work, we put our interest on the MATH domain of the E3-Ligase adaptor protein SPOP to decipher the molecular features underlying its interaction with three different peptides mimicking its physiological partners, the phosphatase Puc, the chromatin component MacroH2A, and the dual-specificity phosphatase PTEN, and also the inhibition of such interactions. Indeed, mutations in the MATH domain of SPOP are major causes of prostate and endometrial cancers. In addition, SPOP is overexpressed in kidney cancer. MATH domain recognizes specific short linear motifs on their substrates to mediate their ubiquitination; thus, targeting such interactions is an interesting therapeutical strategy for developing new anticancer drugs. By employing stopped-flow kinetic binding experiments and extensive site-directed mutagenesis, we addressed the role of specific residues, some of which govern these transient interactions far from the binding site. Our findings are compatible with a scenario in which the binding of the MATH domain with its substrate is characterized by a fine, energetic network regulating its interactions with different ligands. Using peptides computationally designed by our collaborator, targeting MATH interactions, we assessed the inhibition of such interaction. Results are briefly discussed in previous work on the MATH domain. Keywords: PPIs, MATH domain, SPOP, SLIMs, binding, stopped-flow, folding, inhibitors, site-directed mutagenesis.