Prion Fibrillization Is Mediated by a Native Structural Element That Comprises Helices H2 and H3

Abstract

Aggregation and misfolding of the prion protein (PrP) are thought to be the cause of a family of lethal neurodegenerative diseases affecting humans and other animals. Although the structures of PrP from several species have been solved, still little is known about the mechanisms that lead to the misfolded species. Here, we show that the region of PrP comprising the hairpin formed by the helices H2 and H3 is a stable independently folded unit able to retain its secondary and tertiary structure also in the absence of the rest of the sequence. We also prove that the isolated H2H3 is highly fibrillogenic and forms amyloid fibers morphologically similar to those obtained for the full-length protein. Fibrillization of H2H3 but not of full-length PrP is concomitant with formation of aggregates. These observations suggest a "banana-peeling" mechanism for misfolding of PrP in which H2H3 is the aggregation seed that needs to be first exposed to promote conversion from a helical to a ␤-rich structure.Transmissible spongiform encephalopathies are fatal neurodegenerative pathologies that affect humans as well as several other mammalian species. They are thought to be caused by the aggregation and misfolding of the prion protein (PrP).7 According to the "protein-only" hypothesis (1-3), PrP undergoes an ␣-to-␤ transition from its native state (PrP c ) to a misfolded species (PrP sc ), which is believed to act as a template to "infect" and misfold other PrP copies. As in other misfolding pathologies such as Alzheimer and Parkinson diseases, the neurotoxicity of PrP Sc is thought to be associated to an oligomeric form of the protein rather than to the mature aggregates (4).One of the crucial questions that remains unanswered concerns which region(s) of PrP promotes the polymerization process; this information would be both the key for understanding cross-species infectivity and help in decoding the bases of the aggregation process. Different regions have been proposed to be the fibrillogenic seed. PrP c consists of an unstructured N-terminal tail and a folded C-terminal domain formed by three helices (H1, H2, and H3) and a short-stranded ␤-sheet (formed by S1 and S2). H2 and H3 are connected through a disulfide bridge (5). A common view suggests the S1H1S2 region is crucial for ␤-sheet seeding and PrP Sc formation (6, 7). H1 has been implicated as a primary interaction site between PrP Sc and PrP c (8, 9), whereas the loop between S2 and H2, a rigid loop stabilized by its long range interactions with H3 (10), and the C terminus of H3 has been suggested to be recognized by a "Protein-X" that would affect the conversion of PrP c into PrP Sc (11). A study based on intrachain distance estimation performed on tagged PrP amyloid fibrils obtained under chaotropic treatment suggests the involvement of the H2H3 domain of PrP in amyloid formation (12). H/D exchange studies of the amyloid fibrils from human PrP reveal that the ␤-sheet core of PrP amyloids is formed by H2, the major part of H3, and the loop between them (13, 14).We have fol...

Dual Conformation of H2H3 Domain of Prion Protein in Mammalian Cells

“…This is supported by our previous work in vitro showing that the absence of S1-H1-S2 favors fibrillization of H2H3, which occurs more easily than in fulllength PrP (23). Interestingly, it is usual for recombinant proteins such as Sup35, Ure2, or PrP to produce amyloid fibrils in mild denaturing conditions, but fibrillization from recombinant H2H3 does not require these facilitating conditions (23): this is consistent with the spontaneous generation of misfolded H2H3 reported here and implies that the cellular environment is able per se to produce sufficient energetic and thermodynamic fluctuations for H2H3 initial conversion. We thus propose that the separation of S1-H1-S2 from the H2H3 bundle in PrP is a prerequisite for misfolding as suggested in Ref.…”

“…The presence of these post-translational modifications constitutes an indirect proof that the expressed H2H3 was correctly folded and localized according to its signal peptide. This is consistent with H2H3 being an independent folding domain, as proven by NMR studies of the isolated recombinant H2H3 domain (23). This observation is not trivial because H2H3 in PrP is structurally closely embedded in the rest of the globular domain, comprising S1-H1-S2.…”

“…The C-terminal Two Helices H2 and H3 Can Be Stably Transfected in Mammalian Cells-We previously showed by NMR that recombinant ovine H2H3 naturally acquires the same structure as the native H2H3 in full-length PrP (23). We first verified whether the murine sequence is also able to generate an ␣-helical recombinant H2H3.…”

“…Unexpectedly, we observed that there was no natural propensity for the primary sequence of H2H3 to fold into helices and that the disulfide bond and the rest of the proteins are essential in its stabilization (23). It is not unusual for globular proteins to form amyloids (26).…”

“…As shown by the apparent contradiction between these models, a consensus has yet to be found that should be based on in vivo studies. In favor of the H2H3 hypothesis, we have previously shown that (i) H2H3 can be independently generated in an Escherichia coli system and retain its native secondary and tertiary structure (23), (ii) H2H3 is able to fibrillize and reproduce the oligomerization pathways of PrP (23,24), and (iii) in a cellular model of prion infection, polylysines are able to eliminate PK-resistant PrP through a direct interaction with H2H3 (25). These results led to the hypothesis that H2H3 could be the minimal region involved in the change of conformation of PrP.…”