Steric Zipper of the Amyloid Fibrils Formed by Residues 109–122 of the Syrian Hamster Prion Protein

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Abstract

We report the results of atomic force microscopy, Fourier-transform infrared spectroscopy, solid-state nuclear magnetic resonance, and molecular dynamics (MD) calculations for amyloid fibrils formed by residues 109–122 of the Syrian hamster prion protein (H1). Our data reveal that H1 fibrils contain no more than two β-sheet layers. The peptide strands of H1 fibrils are antiparallel with the A117 residues aligned to form a linear chain in the direction of the fibril axis. The molecular structure of the H1 fibrils, which adopts the motif of steric zipper, is highly uniform in the region of the palindrome sequence AGAAAAGA. The closest distance between the two adjacent β-sheet layers is found to be about 5 Å. The structural features of the molecular model of H1 fibrils obtained by MD simulations are consistent with the experimental results. Overall, our solid-state NMR and MD simulation data indicate that a steric zipper, which was first observed in the crystals of fibril-forming peptides, can be formed in H1 fibrils near the region of the palindrome sequence.

Introduction

Prions, defined as proteinaceous infectious particles,1 were first introduced in 1982 to describe a new type of infectious pathogens that propagate through multiple conformers of a normal cellular protein.2 Prions are composed of a modified isoform of the cellular prion protein (PrPC), which is a constituent of normal mammalian cells. This disease-causing isoform is subsequently known as the scrapie prion protein (PrPSc). There are many fatal neurodegenerative diseases closely related to prions such as the Creutzfeldt–Jakob disease and bovine spongiform encephalopathy (commonly known as the “mad cow” disease). The molecular structures of PrPC and some of its variants have been solved by solution-state NMR.3, 4 On the other hand, purified PrPSc is insoluble and its molecular structure is currently not available. It is nowadays a consensus that the amyloidogenic region from residues 90 to 143 plays a significant role in the conversion from PrPC to PrPSc.5 Indeed, it has been shown that the amyloid fibrils formed by the peptide fragments of the mouse PrP89–143 can induce prion disease in transgenic mice.6 Furthermore, secondary-structure analysis indicated that PrPC contains four α-helices, designated H1–H4.5, 7 Synthetic peptides encompassing the H1 region (109–122) have shown to be highly fibrillogenic and toxic to neurons in vitro,8, 9, 10, 11 and they have been used to study prion transmission barrier in vitro.12 Therefore, amyloid fibrils formed by H1 peptides are important biological models for the study of prions. Research studies of amyloid fibrils are being actively pursued because of their occurrence in neurodegenerative diseases.13 In particular, solid-state NMR (SSNMR) spectroscopy has been established as a powerful experimental technique for the structural determination of amyloid fibrils.14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37

Amyloid fibrils formed by peptides of the H1 region have recently been studied by NMR-detected hydrogen–deuterium exchange measurements and Fourier-transform infrared (FT-IR) spectroscopy,38, 39, 40, 41, 42 which provide valuable structural information concerning the overall organization of the β-sheet structures. However, site-specific structural constraints remain unavailable for this class of amyloid fibrils. In this work, atomic force microscopy (AFM), FT-IR, SSNMR spectroscopy, and molecular dynamics (MD) simulations have been applied to elucidate the structure of the amyloid fibrils formed by residues 109–122 of the Syrian hamster prion protein H1 peptides (Ac-MKHMAGAAAAGAVV-NH2) at site-specific level. The backbone torsion ψ angles and the distances between two stacking β-sheet layers determined by SSNMR are in favorable agreement with those obtained from MD simulations. The results show that the molecular structure of the H1 fibrils adopt the steric-zipper motif recently proposed by Sawaya et al.43

Section snippets

Fibril morphology

The effect of the imaging forces on the height of topographic features was examined for the fibril samples (Fig. 1). The results suggest that the fibrils are deformed if the tip force is stronger than 10 nN. The heights of the prion fibrils appeared constant when the intermittent vertical force was smaller than 10 nN. Therefore, the working force in this study was carefully maintained under 10 nN. Figure 2a displays a typical AFM image verifying that, after 1-day incubation at 37 °C, the H1

Backbone torsion ψ angle determination

Backbone torsion angles can be accurately estimated by measuring the 13C and 15N secondary chemical shifts on the basis of the TALOS approach.57 However, this empirical approach is not very useful for the characterization of secondary structures such as a bend or turn, which has been shown to be an important motif in amyloid fibrils.19, 32 There are many different SSNMR techniques suggested for the direct determination of peptide backbone torsion angles ϕ and ψ under MAS.58, 59, 60, 61, 62 Most

Conclusion

Structural determination of steric zipper by SSNMR method is not straightforward, particularly when the distance between two β-sheet layers is comparable to the interstrand distance within the individual β-sheet layer. We show that isotope-edited FT-IR measurements can be carried out for solid fibril samples and provide a very useful structural constraint on which the SSNMR data can be unequivocally analyzed. Our experimental and MD simulation data indicate that a steric zipper, which was first

Sample preparation and characterization

All the chemicals were obtained from NovaBiochem unless stated otherwise. H1 peptides, with the sequence Ac-MKHMAGAAAAGAVV-NH2, were synthesized on a PS3 (Rainin) peptide synthesizer, using a Rink amide resin (0.6 meq/g substitution level; Novabiochem), and 9-fluorenylmethoxycarbonyl chemistry with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation. The synthesis scale was 0.1 mmol, with a fivefold excess and a 2-h coupling time for each unlabeled amino acid,

Acknowledgements

This work was supported by grants from the National Science Council and the Ministry of Education. We thank the anonymous reviewers for their helpful comments.

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