Characterizing the role of the PrPC N-terminal domain in protein and metal binding: NMR and XAS studies

The conversion of the cellular prion protein PrPC into the infectious isoform (PrPSc) is the key event in prion diseases. The physiological role of PrPC remains one of main challenges in prion biology, and it is an absolute requirement also for understanding prion diseases. Putative roles for PrPC are based on its localization in the central and peripheral nervous systems and on PrPC- interacting molecules or metal ions through its unstructured N-terminal domain. We analysed the function of the cellular prion protein using structural biology techniques aimed to analyze the interaction between PrPC and NCAM and PrPC with copper ions. We first focused on the structural determinants responsible for human PrPC (HuPrP) and NCAM interaction using Stimulated Emission Depletion (STED) nanoscopy, surface plasma resonance (SPR) and NMR spectroscopy approaches. Such structural and biological investigations revealed surface interacting epitopes governing the interaction between HuPrP N-terminus and the second module of NCAM Fibronectin type-3 domain, providing molecular details about the interaction between HuPrP and NCAM Fibronectin domain, and revealed a new role of PrPC N- terminus as a dynamic and functional element responsible for protein-protein interaction. Subsequently, we have investigated the role of copper in prion conversion and susceptibility with a special focus on the non-OR copper binding site. The molecular mechanisms of prion conversion are still debated. NMR-based studies on HuPrP and MoPrP globular domains have identified the β2-α2 loop as important element able to modulate the susceptibility of a given species to prion disease. However, recent studies have highlighted also the importance of the N- terminal region in promoting structural rearrangements to PrPSc. We studied copper coordination in the non-OR region of different species including human, sheep, bank vole and opossum. By using in vitro approaches, cell-based and computational techniques, we propose two types of copper coordination geometries, where the type-1 Cu(II) coordination displays a closed non-OR region conformation associated with less-susceptible species, while in type-2 a less structured and solvent exposed non-OR region might render the overall PrPC structure more flexible, therefore we correlate this with higher susceptibility to prion diseases. Our data highlighted how copper coordination in the non-OR copper binding site may explain the different susceptibility to prion diseases observed in these mammalian species. Ultimately, in the present thesis we expanded our knowledge on how the N-terminus of PrPC regulates the physiological functions of PrPC and how it is involved in the prion conversion.