Structural Characterization of Prion Protein Misfolding using Proteins from Resistant Species and Antibodies

Transmissible spongiform encephalopathies are a group of infectious and currently untreatable neurological diseases of humans and farmed and wild animals, that are caused by a unique pathogenic mechanism – misfolding of the cellular prion protein (PrP). PrP is primarily expressed in the nervous system. It is highly conserved among mammals and has orthologs in other vertebrates. The mature PrP is a GPI-anchored glycoprotein attached to the outer leaflet of the plasma membrane. It has two main structural domains. The N-terminal half is flexible and contains four or more metal binding sites. The C-terminal half is folded into a unique prion fold composed of two short β-strands and three α-helices. The misfolding of PrP leads to a poorly understood and aggregation-prone prion species rich in β-sheets. A number of pathologic mutations have been identified, mostly in the C-terminal domain. Some species, such as horses or chicken are considered resistant to prion diseases. To further our understanding of PrP’s pathologic conversion I explored two main areas. Firstly, I determined the crystal structure of the C-terminal, folded domains of horse PrP at 1.08 Å and of chicken PrP at 1.7 Å resolution. Both structures show a typical prion fold and a disordered highly conserved helix-2 helix-3 loop which was structured in crystal structures of PrPs from other species. Secondly, I showed that the β-sheet-rich octameric PrP induced by intermediate urea concentrations at low pH is stable at least for 80 days upon urea removal and is recognized by several antibodies. I determined the crystal structure of PRB7 Fab, a putative β-PrP-specific antibody. This antibody however did not show any binding to PrP or its proposed epitope peptide in the tested conditions. Antibodies D18, P and AMF-1c-120 showed a highly pH-dependant binding to β-octamers. While D18 Fab did not bind β-octamers at low pH, P Fab formed a stoichiometric 1:1 complex with β-PrP octamer, even at pH 4.5. This thesis describes structural features of PrP that may contribute to resistance to prion diseases in some species and provides a roadmap for the antibody-mediated crystallization of the misfolded PrP.Ph.D.2021-11-13 00:00:0