Toward the Identification of the Structural Determinants of Prion Conversion

The post-translational conversion of PrPC into the misfolded, pathogenic form PrPSc plays a key role in prion diseases or transmissible spongiform encephalopathies. These disorders are neurodegenerative maladies affecting both human and animals. One of the crucial questions in prion biology is the identification of the regions on PrPC that lead to the conversion process, whereby most alpha-helical motifs are replaced by betasheet secondary structures. In order to gain insights into the structural determinants involved in PrPSc formation we investigated the effect of HuPrPC point mutations linked to genetic form of prion diseases. Pathological point mutations cause spontaneous formation of PrPSc in the brain. Structural studies with HuPrP variants may provide new clues regarding the conversion mechanism, as well as help in the identification of a “hot spot” on PrPC side which may be a possible target for pharmacological intervention. We first evaluated by molecular dynamics simulations the structural facets of three HuPrP variants located in the globular domain: two pathological mutations, Q212P and E200K, linked to Gerstmann-Sträussler–Scheinker (GSS) and familial Creutzfeldt-Jakob disease (fCJD) respectively, and the protective polymorphism E219K. Such simulation-based structural predictions provided preliminary hints on the effects of mutations clustered in the globular HuPrP domain. Subsequently, we carried out structural investigation determining the high-resolution NMR threedimensional (3D) structure of the truncated recHuPrP(90-231) carrying both the fCJD-linked V210I and the GSS-causing Q212P mutations. Moreover, we determined the 3D NMR structure of the E219K polymorphism in order to find the structural basis responsible for its protective effect. Such structural studies led to the preliminary conclusion that the structural disorders of the b2-a2 loop region together with the increased spacing between this loop and the C-terminal part of a3 helix represent key pathological features. This observation raises the possibility that the spontaneous formation of prions might start from the disruption of the hydrophobic core present in the structured HuPrP domain. We then investigated whether the b2-a2 loop region might be exploited to test the effectiveness of drug candidates that would halt prion replication by binding to this epitope. We took advantage of a camel antibody fragment, denoted as nanobody, raised against the b2-a2 loop region of both Mo and HuPrP. We found that the binding of the nanobody to the b2-a2 loop resulted in inhibition of PrPSc replication when the nanobody was added to chronically infected mouse hypothalamic cells and to fibrillization assays. Finally, we have evaluated the effect of the pathological mutations on the N-terminal unstructured domain. We used synchrotron-based X-ray absorption fine structure (XAFS) technique to study the coordination geometries of both Cu2+ and Cu1+ in the Q212P mutant and we compared these findings with the WT. We clearly showed that Q212P mutant causes a dramatic modification on the non-OR copper binding site in the presence of both copper oxidative states (Cu2+ and Cu1+). These findings are a step forward towards showing a structure-function relationship which provides a biological basis for understanding the spontaneous generation of PrPSc in inherited prion diseases.