Conformational Properties of β-PrP

Laszlo L.P Hosszu,Clare R Trevitt,Samantha Jones,Mark Batchelor,David J Scott,Graham S Jackson,John Collinge,Jonathan P Waltho,Anthony R Clarke

doi:10.1074/jbc.m809173200

Laszlo L.P Hosszu, Clare R Trevitt + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m809173200

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Abstract

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a beta-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126-227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

Highlights

Prion diseases, such as Creutzfeldt-Jacob and GerstmannStraussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)2 [1]
The most coherent and general model proposed far is that PrPC fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers [3, 17]
In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrPSc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrPScdependent conversion reactions (18 –20) or through conversion of PrPC in the absence of a PrPSc template [21,22,23,24,25]. The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized

Summary

The abbreviations used are

PrP, prion protein; AUC, analytical ultracentrifugation; CD, circular dichroism; DTT, 1,4-dithiothreitol; PrPC, cellular PrP isoform; PrPSc, pathogenic (scrapie) PrP isoform; PrP91–231, human prion protein (residues 91–231); HSQC, heteronuclear single quantum coherence; TSP, sodium 3-trimethylsilyl-2,2,3,3-(2H4)-propionate. Many of these non-native states display some of the characteristics of PrPSc, such as increased ␤-sheet content, protease resistance, and a propensity for oligomerization [28, 29, 31] and some have been claimed to be associated with the disease process [34] One such PrP folding intermediate, termed ␤-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native ␣-helical conformation (here termed ␣-PrP), at acidic pH in a reduced state, with the disulfide bond broken [22, 35]. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined ␤-sheet secondary structure, and in conjunction with the NMR data, indicated that ␤-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate [22] Such states have been proposed to be important in amyloid and fibril formation [41]. We use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from ␤-PrP

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION