Prion Domain of Yeast Ure2 Protein Adopts a Completely Disordered Structure: A Solid-Support EPR Study

Sam Ngo,Linh Le,Vicky Chiang,Elaine Ho,Zhefeng Guo,Salvador Ventura

doi:10.1371/journal.pone.0047248

Abstract

Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer’s, Parkinson’s, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer’s amyloid-β monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.

Highlights

Formation of amyloid fibrils and oligomeric intermediates is associated with a wide range of human disorders, including Alzheimer’s disease, prion diseases, and type II diabetes [1]
Our results show that the site-specific residue mobility profile is similar between 7 M guanidine hydrochloride (GdnHCl) and phosphate buffered saline (PBS), suggesting that Ure2p prion domain is completely disordered under native conditions
We have previously shown that Ure2p1– 89-M forms amyloid fibrils that are similar to the isolated Ure2p prion domain [24]

Summary

Introduction

Formation of amyloid fibrils and oligomeric intermediates is associated with a wide range of human disorders, including Alzheimer’s disease, prion diseases, and type II diabetes [1]. The amyloid fibrils involved in different diseases share common characteristics such as cross-b X-ray diffraction pattern, and binding to amyloid-specific dyes thioflavin T and Congo red, different amyloid proteins are distinct in their amino acid sequences and native structures. These amyloid proteins can be divided into two groups: one group with a folded structure in their native soluble state, and the other group that is intrinsically disordered. One example of such study is the binding-induced protein folding within the intrinsically disordered domain of the measles virus nucleoprotein, in which random coil to helix transition and a transiently populated folded state were detected with EPR [7]. We show that a structured state of amyloid-b (Ab) monomer can be detected by EPR [8]

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Results

Conclusion