Mercurio, Flavia Anna (2013) NMR STRUCTURAL AND BINDING STUDIES OF RECOMBINANT PROTEINS OF BIOTECHNOLOGICAL INTEREST. [Tesi di dottorato]

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Abstract

My PhD thesis has been focused on studies carried out by different experimental and computational techniques, such as NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), ITC (Isothermal Titration Calorimetry), molecular docking, mutagenesis, of Sam (Sterile alpha motif) domains containing proteins that play important roles in physiological or pathological processes. Protein-protein interactions are essential for the assembly, regulation, and localization of functional protein complexes in the cell. The analysis of these interactions at molecular level is of great interest in many biotechnological fields, as it provides useful information for the design of molecules able to mimic the binding sites and thus, presenting potential therapeutic applications. Protein-protein associations are mediated by specific domains, such as Sam domains. Within this thesis two heterotypic Sam-Sam interactions were studied in details: 1) the EphA2-Sam/Odin-Sam1 complex; 2) the Odin-Sam1/Arap3-Sam association. The tyrosine kinase receptor EphA2 plays a fundamental role in tumorigenesis. The process of EphA2 endocytosis and consequent degradation has been investigated as potential route to reduce tumor malignancy. Odin belongs to the ANKS (Ankyrin repeat domain containing and Sam) protein family; it contains two Sam domains in tandem (Sam1 and Sam2), and is able to regulate EphA2 receptor endocytosis. Instead, Arap3 (Arf GAP, Rho GAP, Ankyrin repeat and PH domains) is a protein involved in the phosphoinositol-3-kinase (PI3K) signaling pathways, which regulates biological processes connected to cell motility. Firstly, by multidimensional (2D and 3D) NMR methods, the solution structure of Odin-Sam1 was determined. It consists of five -helices and represents a canonical Sam domain fold. Afterwards, binding studies with EphA2-Sam, and Arap3-Sam were conducted. SPR and ITC experiments revealed a low micromolar binding affinity of Odin-Sam1 for both EphA2-Sam and Arap3-Sam. The reciprocal interaction surfaces of these Sam domains were identified by NMR chemical shift perturbation experiments, and 3D models of the complexes were built by molecular docking techniques. These studies suggested that Odin-Sam1/EphA2-Sam and Odin-Sam1/Arap3-Sam complexes might adopt the canonical Sam-Sam interaction topology called "Mid-Loop/End-Helix", where the central portion of Odin-Sam1 and the C-terminal helix of either Arap3-Sam and EphA2-Sam contribute the binding surfaces. A peptide, named Sam3, which encompasses the central portion of the Odin-Sam1 involved in complexes formation with EphA2-Sam and Arap3-Sam, together with its C-terminal helix, was synthesized and analyzed by 2D NMR techniques. The peptide reveals unstructured in phosphate buffer, whereas it shows propensity to adopt more ordered helical structures in water/trifluoroethanol mixtures. Based on the new structural insights gained within my thesis, libraries of molecules (peptides and peptido-mimetics), that could selectively interfere with Odin-Sam1/EphA2-Sam or Odin-Sam1/Arap3-Sam associations, and prove useful in therapeutic and diagnostic applications, will be designed in the near future.

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