Introduction of glutamines into the B2-H2 loop promotes prion protein conversion.

In prion diseases cellular prion protein (PrP(C)) undergoes conformational transition into the ß-sheet-rich form (PrP(Sc)). PrP(C) consists of the disordered N-terminal part and a C-terminal globular domain containing three α-helices (H1, H2, H3) and an antiparallel beta sheet (B1, B2). B2-H2 loop, which has a focal role in the species barrier, contains the highest density of asparagine (N) and glutamine (Q) residues in the whole sequence. Q/N-rich domains are essential for the conversion of yeast prions. We investigated the role of Q/N residues in the B2-H2 loop in PrP conversion. We prepared mouse PrP mutants with increasing number of consecutive Q/N residues in the B2-H2 loop. Stability of the mutants decreased with the increasing number of inserted glutamines. In vitro conversion of mutants yielded fibrils of similar morphology as the wild-type PrP. Q/N mutants accelerated fibrillization in comparison to the wild-type PrP, with mutant containing the most glutamines having the shortest lag phase. The effect of Q/N residues was specific for the B2-H2 loop and was not due to simple increase in flexibility as the introduction of Gly-Ser or Ala residues slowed the conversion despite their decreased stability. Our results thus suggest that Q/N residues in the B2-H2 loop of PrP promote protein conversion and may represent a link to conversion of Q/N-rich prions.

Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion.

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the ß-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.