Probing PrP^Sc Structure Using Chemical Cross-Linking and Mass Spectrometry:  Evidence of the Proximity of Gly90 Amino Termini in the PrP 27−30 Aggregate

Abstract

Elucidation of the structure of PrP Sc continues to be one of the most important and difficult challenges in prion research. This task, essential for gaining an understanding of the basis of prion infectivity, has been hampered by the insoluble, aggregated nature of this molecule. We used a combination of chemical cross-linking, proteolytic digestion, and mass spectrometry (MALDI-TOF and nanoLC-ESI-QqTOF), in an attempt to gain structural information about PrP 27-30 purified from the brains of Syrian hamsters infected with scrapie. The rationale of this approach is to identify pairs of specific amino acid residues that are close enough to each other to react with a bifunctional reagent of a given chain length. We cross-linked PrP 27-30 with the amino-specific reagent bis(sulfosuccinimidyl) suberate (BS 3 ), obtaining dimers, trimers, and higher-order oligomers that were separated by SDS-PAGE. In-gel digestion followed by mass spectrometric analysis showed that BS 3 reacted preferentially with Gly90. A cross-link involving two Gly90 amino termini was found in cross-linked PrP 27-30 dimers, but not in intramolecularly crosslinked monomers or control samples. This observation indicates the spatial proximity of Gly90 amino termini in PrP 27-30 fibrils. The Gly90-Gly90 cross-link is consistent with a recent model of PrP 27-30, based on electron crystallographic data, featuring a fiber composed of stacked trimers of PrP monomers; specifically, it is compatible with cross-linking of monomers stacked vertically along the fiber axis but not those adjacent to each other horizontally in the trimeric building block. Our results constitute the first measured distance constraint in PrP Sc .Prions were originally defined as "proteinaceous infectious particles", and very substantial and diverse evidence has accumulated to suggest that, indeed, their only component might be an intrinsically infectious protein (2). Transmissible spongiform encephalopathies (TSEs), 1 a group of fatal neurodegenerative diseases affecting humans and animals, are associated with a prion, termed PrP (prion protein) (2-6). PrP can exhibit two conformations, PrP C and PrP Sc . The latter is associated with TSEs, and PrP showing the physicochemical characteristics of PrP Sc is isolated as the main and most probably only component of the TSE infectious agent. PrP C can convert into PrP Sc in the presence of preformed PrP Sc , through a poorly understood molecular process (6, 7). The structure of PrP C has been characterized by NMR (8), but that of PrP Sc is largely unknown, as its insolubility in nondenaturing solvents has seriously hampered analytical efforts. It is known, however, that PrP Sc and PrP C differ with respect to secondary, tertiary, and quaternary structure (3,9). No covalent differences have been detected between the two molecules (10), although the possibility that post-translational modifications of a small set of PrP C molecules could trigger structural changes relevant to initiation of conversion to PrP Sc cannot be ruled out...

Parallel In-register Intermolecular β-Sheet Architectures for Prion-seeded Prion Protein (PrP) Amyloids

“…Res is provided in a study by Onisko et al (64) in which dimers and trimers cross-linked via Gly-90 with bis(sulfosuccinimidyl) suberate (BS 3 ) were observed. A stacked in-register alignment of the N terminus would space Gly-90 at ϳ4.8 or 9.6 Å between dimers or trimers, respectively.…”

“…Such dimers and trimers would be susceptible to cross-linking by BS 3 , which has a maximum cross-linking distance of 11.4 Å. In contrast, four or more stacked monomers would be spaced at a distance greater than 14.4 Å and would therefore be outside the cross-linking range of BS 3 ; accordingly, no cross-linked tetramers were observed (64). Significantly, these findings would also be incompatible with some of the current models in which the axial spacing between the same residues on adjacent monomers would be greater than 14.4 Å.…”

A Structural Overview of the Vertebrate Prion Proteins

“…Chemical cross-linking experiments support this model. 119 A b-helix is however inconsistent with mass spectrometry and with H/D backbone amide exchange data obtained from fibrillar samples of the recombinant protein 120 which suggest that the sequence covering residues ~169-221 contributes to the core of the b-sheet, whereas residues 90-168 do not form H-bonded structures. A way to explain these differences is to assume that they arise from the different nature of the specimens used.…”

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS

“…PK digestion of the 263K strain of PrP Sc cleaves the amino terminal domain to give an infectious core consisting primarily of 142 amino acids with an amino terminal Gly90 that is referred to as PrP 27-30 [2].Å IfÅ theÅ PKÅ digestionÅ ofÅ PrPÅ Sc is done in the presence of detergents, rod-shaped particles are obtained, composed of monomers of PrP 27-30. By use of chemical cross-linking and mass spectrometry, the amino terminal Gly90 residues of adjacent monomers in PrP 27-30 rods have been shown to be locatedÅ withinÅ 11Å ÅÅ ofÅ eachÅ otherÅ [11].…”