New insights into the molecular mechanism of amyloid formation from cysteine scanning

Abstract

Our laboratory recently reported the identification of a peptide region, QVNI, within the prion domain of the yeast protein Ure2 that may act as an initiation point for fibril formation.1 This potential amyloid-forming region, which corresponds to residues 18-21 of Ure2, was initially identified by systematic cysteine scanning of the Ure2 prion domain. The point mutant R17C, and the corresponding octapeptide CQVNIGNR, were found to form fibrils rapidly under oxidative conditions due to the formation of a disulfide bond. Deletions within the QVNI sequence cause the fibril formation ability of R17C Ure2 to be lost. The aggregation propensity of this region is strongly modulated by its preceding residue: replacement of R17 with a hydrophobic residue promotes fibril formation in both full-length Ure2 and in the corresponding octapeptides. The wild-type octapeptide, RQVNIGNR, also forms fibrils, and is the shortest amyloid-forming peptide found for Ure2 to date. Interestingly, the wild-type octapeptide crystallizes readily and so provides a starting point towards obtaining high resolution structural information for the amyloid core of Ure2 fibrils.

Acknowledgements

Work in the Perrett laboratory is supported by grants from the National Natural Science Foundation of China (30620130109, 30670428, 30870482), the Chinese Ministry of Science and Technology (2006CB500703, 2006CB910903) and the Chinese Academy of Sciences (KSCX2-YW-R-119). We thank members of the Perrett laboratory for critical reading of the manuscript.

Figures and Tables

Figure 1 The prion domain sequence of Ure2. The Gln/Asn-rich region of the 354-residue Ure2 protein extends from residues 1–89.43 The residues mutated to Cys and tested for amyloid-forming ability under oxidizing conditions1 are underlined. R17 is shown in bold.

Figure 2 Crystals of the wild-type Ure2 octapeptide RQVNINIGNRNR viewed under the polarized microscope. The crystals were formed by incubating the octapeptide solution (10 mg/ml in Tris buffer pH 8.4 with 200 mM NaCl) at 4°C overnight.