Prion infection is characterized by the conversion of host cellular prion protein (PrP C ) into disease-related conformers (PrP Sc ) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for PrP C rather than PrP Sc . We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for PrP C and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native PrP C conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular ␤-sheet. The importance of this residue in mediating protein-protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease.Creutzfeldt-Jakob disease ͉ PrP-Fab complex ͉ monoclonal antibody ͉ prion therapeutics

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, unphysiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening identified syntaxin 6 (Stx6) as a risk factor for Creutzfeldt-Jakob disease. Analysis of the protein in NAA revealed that Stx6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at a highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, Stx6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that PrP formed less-ordered aggregates with Stx6 instead of highly ordered fibrils, which contained Stx6. These data strongly suggest that the protein directly alters PrP self-assembly and, uniquely, acts as an 'anti-chaperone', which promotes toxic aggregation intermediates by inhibiting fibril formation.