Prion protein amyloid formation under native-like conditions involves refolding of the C-terminal α-helical domain

Abstract

Transmissible spongiform encephalopathies are associated with conformational conversion of the cellular prion protein, PrPC, into a proteinase K-resistant, amyloid-like aggregate, PrPSc. Although the structure of PrPSc remains enigmatic, recent studies have afforded increasingly detailed characterization of recombinant PrP amyloid. However, all previous studies were performed using amyloid fibrils formed in the presence of denaturing agents that significantly alter the folding state(s) of the precursor monomer. Here we report that PrP amyloid can also be generated under physiologically relevant conditions, where the monomeric protein is natively folded. Remarkably, site-directed spin labeling studies reveal that these fibrils possess a β-core structure nearly indistinguishable from that of amyloid grown under denaturing conditions, where the C-terminal α-helical domain of the PrP monomer undergoes major refolding to a parallel and in-register β-structure upon conversion. The structural similarity of fibrils formed under drastically different conditions strongly suggests that the common β-sheet architecture within the ∼160-220 core region represents a distinct global minimum in the PrP conversion free energy landscape. We also show that the N-terminal region of fibrillar PrP displays conformational plasticity, undergoing a reversible structural transition with an apparent pKa of ∼5.3. The C-terminal region, on the other hand, retains its β-structure over the pH range 1-11, whereas more alkaline buffer conditions denature the fibrils into constituent PrP monomers. This profile of pH-dependent stability is reminiscent of the behavior of brain-derived PrP Sc, suggesting a substantial degree of structural similarity within the β-core region of these PrP aggregates. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.