Probing the Structure of the Infectious Amyloid Form of the Prion-forming Domain of HET-s Using High Resolution Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Alexis Nazabal,Marie-Lise Maddelein,Marc Bonneu,Sven J Saupe,Jean-Marie Schmitter

doi:10.1074/jbc.m413185200

Abstract

The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218-289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218-289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218-289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218-289) sequence. The HET-s-(218-289) fibrils are thus likely to be organized as a succession of beta-sheet segments interrupted by short turns or short loops.

Highlights

Amyloids are fibrillar protein aggregates composed of a “cross-␤” structure in which ␤-strands are oriented perpendicular to the fiber axis
We have previously shown that HET-s fibrils submitted to proteinase K digestion retain infectivity (21)
It remained possible that infectivity of the proteinase K digested HET-s amyloids could be due to an undigested contaminating fraction of full-length protein or else that the HET-s-(218 –289) domain adopts a different structure in full-length HET-s amyloid as in amyloids formed from the HET-s-(218 –289) peptide

Summary

Introduction

Amyloids are fibrillar protein aggregates composed of a “cross-␤” structure in which ␤-strands are oriented perpendicular to the fiber axis. The best characterized prion models are the yeast Ure2p and Sup35p proteins and the HET-s protein from the fungus Podospora anserina (14). All of these proteins form amyloid fibrils in vitro (15–18). The HET-s prion protein of P. anserina is involved in a genetically programmed cell death reaction termed heterokaryon incompatibility. Hydrogen/deuterium exchange combined with mass spectrometry (HXMS) has become a powerful tool for the study of protein structures and dynamics (3, 23–27). More details concerning HXMS methodology are presented in a review by Engen and Smith (25)

Methods

Results

Conclusion