The conversion of the prion protein (PrPC) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrPC interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrPC constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrPC coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

Abstract-Prion diseases are fatal neurodegenerative disorders linked to the deposition of the abnormal prion protein isoform called PrPSc or prion. The key molecular events triggering the diseases are the conformational changes from the normal cellular α-helical prion protein PrPC to the pathological β-sheet enriched PrPSc. Therefore, understanding the mechanism and factors underlying the conversion process is essential to find possible diagnostic tools and treatments. Copper has long been known to correlate with neurodegenerative dysfunctions; PrPC is a copper binding protein via histidine residues in the highly conserved octapeptide repeats (OR) and the non-OR region located in the disordered N-terminal tail of the protein. The role of copper in facilitating protein aggregation and disease progression remains elusive. This study describes the impact of histidine residues on prion replication. By analyzing mouse PrP constructs that carry artificial mutations at histidines in the OR and non-OR, we provide cell evidence for the critical role of the non-OR copper binding site at histidine 95 in prion conversion. We also contribute to better understanding of the mechanisms and primary sites for prion conversion and replication. Our findings establish a platform for further studies aimed at elucidating the role of the H95 mutant in de novo prion diseases when expressed in transgenic mice.