Prion Protein Amyloid Formation under Native-like Conditions Involves Refolding of the C-terminal α-Helical Domain

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 PrPSc, suggesting a substantial degree of structural similarity within the β-core region of these PrP aggregates. 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 PrPSc, suggesting a substantial degree of structural similarity within the β-core region of these PrP aggregates. Transmissible spongiform encephalopathies (TSEs), 3The abbreviations used are: TSE, transmissible spongiform encephalopathies; PrPC, cellular prion protein; huPrP, human prion protein; PrPSc, disease-associated, proteinase K-resistant prion protein isoform; rPrP, recombinant prion protein; rPrPAm, recombinant prion protein amyloid; rPrPAmN, recombinant prion protein amyloid formed under native conditions; rPrPAmU, recombinant prion protein amyloid formed under denaturing conditions; PK, proteinase K; GdnHCl, guanidine hydrochloride; ThT, thioflavin T; EPR, electron paramagnetic resonance; SDSL, site-directed spin labeling. or prion diseases, are a group of fatal neurodegenerative disorders encompassing bovine spongiform encephalopathy in cattle, scrapie in sheep, chronic wasting disease in cervids, and Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia, and kuru in humans (1.Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4106) Google Scholar, 2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 3.Caughey B. Baron G.S. Nature. 2006; 443: 803-810Crossref PubMed Scopus (247) Google Scholar, 4.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1110) Google Scholar, 5.Aguzzi A. Polymenidou M. Cell. 2004; 116: 313-327Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 6.Weissmann C. Nat. Rev. Microbiol. 2004; 2: 861-871Crossref PubMed Scopus (319) Google Scholar, 7.Caughey B. Chesebro B. Adv. Virus Res. 2001; 56: 277-311Crossref PubMed Google Scholar). In the case of human TSEs, the disease state may arise either sporadically, via inheritance, or through exposure to infectious prions. In all cases, however, neurodegeneration is intrinsically tied to conformational conversion of the normal cellular prion protein (PrPC) to a misfolded aggregate isoform dubbed PrPSc (1.Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4106) Google Scholar, 2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 3.Caughey B. Baron G.S. Nature. 2006; 443: 803-810Crossref PubMed Scopus (247) Google Scholar, 4.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1110) Google Scholar, 5.Aguzzi A. Polymenidou M. Cell. 2004; 116: 313-327Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 6.Weissmann C. Nat. Rev. Microbiol. 2004; 2: 861-871Crossref PubMed Scopus (319) Google Scholar, 7.Caughey B. Chesebro B. Adv. Virus Res. 2001; 56: 277-311Crossref PubMed Google Scholar). The “protein-only” hypothesis asserts that transmission of TSEs does not require nucleic acid and that the sole infectious agent is PrPSc, which is able to self-propagate by first binding to and then inducing a conformational conversion in PrPC to the PrPSc state (1.Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4106) Google Scholar, 2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar). Although the molecular details of such a mechanism of disease propagation are poorly understood, extensive biochemical and genetic investigations strongly support the principle of protein-based infectivity (1.Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4106) Google Scholar, 2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 3.Caughey B. Baron G.S. Nature. 2006; 443: 803-810Crossref PubMed Scopus (247) Google Scholar, 4.Collinge J. Annu. 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Both PrPC and PrPSc appear to share identical covalent structure but are readily distinguished by various biochemical properties. Cellular PrPC is a 209-residue glycoprotein with a C-terminally attached glycosylphosphatidylinositol anchor (2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 3.Caughey B. Baron G.S. Nature. 2006; 443: 803-810Crossref PubMed Scopus (247) Google Scholar, 4.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1110) Google Scholar, 5.Aguzzi A. Polymenidou M. Cell. 2004; 116: 313-327Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). It consists of a flexible N-terminal region and a primarily α-helical C-terminal domain, stabilized by a disulfide bond (12.Riek S. M. Nature. PubMed Scopus Google Scholar, S.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. C. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In to PrPC, the misfolded PrPSc isoform is aggregate in displays β-sheet to proteinase and in (2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 3.Caughey B. Baron G.S. Nature. 2006; 443: 803-810Crossref PubMed Scopus (247) Google Scholar, 4.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1110) Google Scholar, 5.Aguzzi A. Polymenidou M. Cell. 2004; 116: 313-327Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 6.Weissmann C. Nat. Rev. Microbiol. 2004; 2: 861-871Crossref PubMed Scopus (319) Google Scholar, 7.Caughey B. Chesebro B. Adv. Virus Res. 2001; 56: 277-311Crossref PubMed Google Scholar). to other disorders of protein such and disease B. Annu. Rev. Neurosci. PubMed Scopus Google Scholar, Annu. 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In we have the of amyloid to recombinant prion protein by inducing conformational conversion under more physiologically relevant We that the familial a conversion to amyloid fibrils upon pH in the of where the PrP monomer is to its native α-helical Remarkably, of the conformational state of the precursor amyloid fibrils with parallel and in-register β-structure common molecular architecture to a distinct global free energy minimum in the folding of PrP human prion protein used in were and with the spin labeling to W.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: PubMed Scopus Google Scholar). the were in pH and of of amyloid fibrils under conditions performed by of protein in pH with under denaturing conditions performed in GdnHCl, pH with and were using a thioflavin and to the presence and of the amyloid fibrils formed A.C. W.K. 2005; PubMed Scopus Google Scholar, E.M. W.K. 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The in of the buffer and into a were using a with the to and were with a of from to on the degree of spin and within were to the of to the of amyloid fibrils were in pH The in the buffer of of the were and the protein of the which from the amyloid using a of were to have on the of in the that is to be The of protein in the upon in GdnHCl, the of monomer to be a of and of amyloid fibrils in a using the pH pH pH pH pH pH pH pH pH of and in of the to the protein in pH were with pH to the pH and of the The of protein in the upon in pH and the pH The protein then be and the of PrP a studies of fibrillar were in the presence of or of different the alkaline conditions a of the free by the However, the of protein and the associated in were by nearly identical by and the of to in the of of amyloid fibrils formed by recombinant PrP in recent and structural of prion protein conversion to in A.C. W.K. 2005; PubMed Scopus Google Scholar, S.B. J. Chem. 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W.K. 2005; PubMed Scopus Google amyloid under these conditions The fibrillar formed by the native precursor appear to be to grown pH in the presence of are the with studies M. J. 2006; PubMed Scopus Google that fibrils are with to of of fibrils formed under native conditions, on the other hand, are more a significantly of fibrils are in whereas fibrils are to from the a with under that fibrillar can be formed from the monomer native rPrP, the remains fibrils formed under such drastically different conditions are the molecular we site-directed spin labeling with of of into the structure of amyloid fibrils W.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: PubMed Scopus Google Scholar, M. S. P. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, M. M. J. J. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). 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Here we a of the in natively formed Remarkably, all within the ∼160–220 region of these we nearly identical to in fibrils the molecular of to be β-core however, were and fibrils and upon of by fibrils using a of and protein of the of these degree of structural However, the degree of to amyloid core encompassing where that on of with parallel and in-register of In to within the ∼160–220 more and fibrils were core region, N-terminal to In these N-terminal and with of within region of in pH of PrP the of structural and we the of pH on fibrils were grown under and denaturing conditions, and of fibrils grown in pH and fibrils grown in pH and in the buffer were these were the pH buffer pH and fibrils pH and were all within the ∼160–220 core region, of were to in pH and and fibrils have formed under different buffer conditions, the core of these fibrils appear to be with In of the β-core region are in spin and these of fibrils in pH buffer in and a in with these of fibrils and of fibrils and where pH in with indistinguishable from of fibrils and This also all other N-terminal to reversible upon of buffer in to the core region, the structure in the N-terminal of amyloid can be by in the pH of the pH-dependent structural a pH performed using fibrils from such a fibrils the is in with the in of pH N-terminal and a transition with an apparent pKa of In within the ∼160–220 region, in were over the pH range and of PrP by the the apparent of the β-core structure to we to and alkaline Remarkably, in the presence of of within the ∼160–220 that the molecular architecture of the core region is under alkaline conditions these by of an PrP monomer that such conditions of amyloid fibrils into constituent monomers. The apparent of by pH-dependent and were using a on the different of fibrillar and monomeric protein under the of in the core the stability of amyloid fibrils grown under different conditions using a with were and by the by in of within the core region these that stability and that of the common amyloid core is associated with into PrP monomers. Although the molecular of the infectious agent the TSEs is in a of that the in these is associated with conformational conversion of monomeric PrPC to proteinase K-resistant, amyloid-like PrPSc (1.Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4106) Google Scholar, 2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 3.Caughey B. Baron G.S. Nature. 2006; 443: 803-810Crossref PubMed Scopus (247) Google Scholar, 4.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1110) Google Scholar, 5.Aguzzi A. Polymenidou M. 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Sci. 2005; PubMed Scopus Google Scholar, W.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: PubMed Scopus Google Scholar, S.B. Science. 2004; PubMed Scopus Google Scholar, W.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: PubMed Scopus Google Scholar, J. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S.B. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the α-helical PrP the remains to the formed in such an are of PrP fibrillar formed under conditions, such Here we report in to conversion under denaturing conditions, with amyloid can be formed by in the of denaturing This which fibrillar to of J. 2005; PubMed Scopus Google represents the core of PrPSc, is to the disease and to be associated with prion disorders (2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 4.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1110) Google Scholar). The conditions in are of cellular such of the of PrPSc in B. J. PubMed Google Scholar, A. S.B. J. Chem. Full Text PDF PubMed Google Scholar). under these conditions the PrP monomer is to be with native α-helical structure of the C-terminal domain, by structural and pH (12.Riek S. M. Nature. PubMed Scopus Google Scholar, S.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. C. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that of the monomeric precursor is a conversion of prion protein to the amyloid Although the amyloid under buffer conditions studies on the mechanism of prion protein the of on the structure of the We that the molecular architecture of amyloid fibrils formed under conditions is to that amyloid fibrils grown in the presence of where of the precursor monomer is P. W.K. J. Chem. Full Text Full Text PDF PubMed Scopus Google In cases, the conversion refolding of the C-terminal and amyloid fibrils possess an identical architecture within the ∼160–220 core region, where the major folding is a parallel and in-register β-structure of monomeric The that of under conditions with different conformational of the precursor monomer to of amyloid fibrils with a common β-core strongly that common architecture is in the acid of and the fibrillar structure a distinct global minimum in the prion protein free energy landscape. The is reminiscent of the where in conversion under native and denaturing conditions in fibrillar with β-core by J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). however, is that the monomer is a primarily β-sheet protein, conversion of PrP refolding of the α-helical domain to the Although the ∼160–220 of PrP to a β-sheet structure upon the conversion to to brain-derived PrPSc, recombinant PrP fibrils are by a significantly core and infectivity A.C. Sci. 2005; PubMed Scopus Google Scholar, S.B. Science. 2004; PubMed Scopus Google Scholar, W.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: PubMed Scopus Google Scholar). studies that the glycosylphosphatidylinositol anchor of PrPC, not in recombinant is the infectivity of the prion agent B. M. C. C. Baron S. B. M. Science. 2005; PubMed Scopus Google Scholar, A. M. M. S.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, C. S.B. J. PubMed Scopus Google Scholar). conformational of PrP and Although in the N-terminal and A.C. Sci. 2005; PubMed Scopus Google the of brain-derived PrPSc is to (2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 6.Weissmann C. Nat. Rev. Microbiol. 2004; 2: 861-871Crossref PubMed Scopus (319) Google Scholar). It is that N-terminal is associated with distinct (2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar). conformational within region to the and PrP aggregate Remarkably, we that the N-terminal of also structural undergoing a pH-dependent structural transition pKa whereas the β-core remains and to the a of the C-terminal ∼160–220 to parallel and in-register we that the structural and PrPSc. recent studies by B. Nat. 2007; PubMed Scopus Google that fibrillar with an within the region can be the conversion is with brain-derived PrPSc. to N-terminal within PrPSc of cellular have to prion protein conversion in (2.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 3.Caughey B. Baron G.S. Nature. 2006; 443: 803-810Crossref PubMed Scopus (247) Google Scholar, Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). Although that such the conversion major refolding of the α-helical we more however, is that these cellular agents in with the conversion of the PrP domain to and the of the N-terminal in an aggregate with and recent by N.R. Harris B.T. Rees J.R. Supattapone S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9741-9746Crossref PubMed Scopus (519) Google suggests the of such have that transmissible can be formed from PrPC with in the presence of The of PrP be to with such a and in N-terminal such nucleic and have in B. Baron G.S. Nature. 2006; 443: 803-810Crossref PubMed Scopus (247) Google Scholar, J. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. Full Text Full Text PDF PubMed Scopus Google such can be to the to an infectious aggregate be that these cellular agents and of the aggregates. from structural the reveal an of to of We that of pH does not in structural within the and fibrils can be into constituent under alkaline Remarkably, these pH are reminiscent of the behavior of brain-derived PrPSc. a common of prion and associated of of PrPSc in S.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar, M. M. S. J. Sci. PubMed Scopus Google a pH to that to of into monomeric PrPSc is to infectivity under the conditions where is to its fibrillar β-structure P. J. PubMed Scopus Google Scholar). is to the of structural prion protein the a substantial degree of structural recombinant PrP amyloid and the infectious PrPSc with