X-ray absorption fine structure studies on human prion protein non-octarepeat copper binding site

The post-translational conversion of PrPC into the misfolded, pathogenic form PrPSc plays a key role in prion diseases or transmissible spongiform encephalopathies. PrPC interacts with metal ions, in particular copper and zinc, through the octarepeat and non-octarepeat binding sites. One of the crucial questions in prion biology is the identification of the regions on PrPC that lead to the conversion process, whereby most α-helical motifs are replaced by β-sheet secondary structures. In order to gain insights into the structural determinants involved in PrPSc formation we investigated the effect of human PrPC (HuPrP) point mutations linked to the genetic form of prion diseases. Pathological point mutations cause spontaneous formation of PrPSc in the brain. We carried out a structural investigation to determine the highresolution NMR three-dimensional 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 β2-α2 loop region, together with the increased spacing between this loop and the C-terminal part of α3 helix are key pathological features. This observation raises the possibility that the spontaneous formation of prions might start with the disruption of the hydrophobic core present in the structured HuPrP domain. We then 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 Zn2+, Cu2+ and Cu+ at two pH values (5.5 and 7) in different HuPrP constructs carrying pathological mutations. We clearly showed that mutations and pH exchanges cause a dramatic modification on the non-OR copper binding site in the presence of metal ions. These findings provide a structure-function relationship that lays a biological basis for understanding the spontaneous generation of PrPSc in inherited prion diseases.