Diversity in prion protein oligomerization pathways results from domain expansion as revealed by hydrogen/deuterium exchange and disulfide linkage

Abstract

The prion protein (PrP) propensity to adopt different structures is a clue to its biological role. PrP oligomers have been previously reported to bear prion infectivity or toxicity and were also found along the pathway of in vitro amyloid formation. In the present report, kinetic and structural analysis of ovine PrP (OvPrP) oligomerization showed that three distinct oligomeric species were formed in parallel, independent kinetic pathways. Only the largest oligomer gave rise to fibrillar structures at high concentration. The refolding of OvPrP into these different oligomers was investigated by analysis of hydrogen/deuterium exchange and introduction of disulfide bonds. These experiments revealed that, before oligomerization, separation of contacts in the globular part (residues 127-234) occurred between the S1-H1-S2 domain (residues 132-167) and the H2-H3 bundle (residues 174 -230), implying a conformational change of the S2-H2 loop (residues 168 -173). The type of oligomer to be formed depended on the site where the expansion of the OvPrP monomer was initiated. Our data bring a detailed insight into the earlier conformational changes during PrP oligomerization and account for the diversity of oligomeric entities. The kinetic and structural mechanisms proposed here might constitute a physicochemical basis of prion strain genesis.folding ͉ kinetics ͉ oligomers ͉ strain

Misfolding of the prion protein: linking biophysical and biological approaches

“…This opening mechanism illustrated in Figure 4c implies a conformational change in the S2-H2 hingeloop corresponding to residues 168-173. Altogether kinetic and structural data reported by Eghiaian et al are consistent with observations related to the PrP C /PrP Sc conversion in vivo [33]. The conformation of the S2-H2 loop seems to play an important role in PrP C /PrP Sc conversion.…”

“…4a) [33]. The kinetic investigation showed that PrP can generate different types of oligomers according to a parallel oligomerization pathway (Fig.…”

Dual Conformation of H2H3 Domain of Prion Protein in Mammalian Cells

“…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. 42 and that exposing or isolating H2H3 is a necessary step in mammalian cells for misfolding and for generation of aggregated PKresistant species.…”

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

“…Furthermore, electron microscopy has detected a head-to-head disulfide-bonded interaction (11). The structure of pathogenic serpin polymers has thus not been unambiguously resolved, although detailed knowledge of the structure of these polymers is critical for understanding the basis of serpin-linked diseases and for designing effective therapeutics.Hydrogen/deuterium exchange measured by mass spectrometry (HXMS) has proven to be a powerful method for probing the structure and dynamics of proteins and protein assemblies that are not accessible to other techniques, and several recent studies have employed HXMS to gain insight into the structural organization of amyloid fibrils (12)(13)(14). HXMS provides information on dynamics and solvent accessibility throughout the entire structure of a protein and does not require the attachment of exogenous labels (15).…”

“…Hydrogen/deuterium exchange measured by mass spectrometry (HXMS) has proven to be a powerful method for probing the structure and dynamics of proteins and protein assemblies that are not accessible to other techniques, and several recent studies have employed HXMS to gain insight into the structural organization of amyloid fibrils (12)(13)(14). HXMS provides information on dynamics and solvent accessibility throughout the entire structure of a protein and does not require the attachment of exogenous labels (15).…”