Intermolecular alignment in Y145Stop human prion protein amyloid fibrils probed by solid-state NMR spectroscopy

Abstract

The Y145Stop mutant of human prion protein, huPrP23-144, has been linked to PrP cerebral amyloid angiopathy, an inherited amyloid disease, and also serves as a valuable in vitro model for investigating the molecular basis of amyloid strains. Prior studies of huPrP23-144 amyloid by magic-angle-spinning (MAS) solid-state NMR spectroscopy revealed a compact β-rich amyloid core region near the C-terminus and an unstructured N-terminal domain. Here, with the focus on understanding the higher-order architecture of huPrP23-144 fibrils, we probed the intermolecular alignment of β-strands within the amyloid core using MAS NMR techniques and fibrils formed from equimolar mixtures of (15)N-labeled protein and (13)C-huPrP23-144 prepared with [1,3-(13)C(2)] or [2-(13)C]glycerol. Numerous intermolecular correlations involving backbone atoms observed in 2D (15)N-(13)C spectra unequivocally suggest an overall parallel in-register alignment of the β-sheet core. Additional experiments that report on intermolecular (15)N-(13)CO and (15)N-(13)Cα dipolar couplings yielded an estimated strand spacing that is within ∼10% of the distances of 4.7-4.8 Å typical for parallel β-sheets.

Figure 1

(A) Partial amino acid sequence of huPrP23-144. Residues exhibiting significant conformational dynamics are shown in red font, and relatively rigid amyloid core residues are shown in black font^, with the three β-strand segments predicted using TALOS+ highlighted by gray rectangles. Also shown is a summary of the intermolecular ¹⁵N-¹³C correlations observed in z-filtered TEDOR spectra recorded for the mixed 1,3-¹³C/¹⁵N- and 2-¹³C/¹⁵N huPrP23-144 fibril samples (panels (D) and (G) and panel (C) of Figure S1). Thick green rectangles under residue i indicate than an unambiguous intermolecular ¹⁵N-¹³C correlation was observed between the amide ¹⁵N of residue i and ¹³CO_i-1 and/or ¹³CO_i (NCO), ¹³Cα_i-1 and/or ¹³Cα_i (NCA), and side-chain ¹³C of residue i (NCX). Narrow green rectangles correspond to ambiguous intermolecular ¹⁵N-¹³C correlations. (B–D) ¹⁵N-¹³CO (NCO) correlation spectra of huPrP23-144 amyloid fibrils recorded at 500 MHz ¹H frequency, 11.111 kHz MAS rate and effective sample temperature of ~5 °C using the z-filtered TEDOR pulse scheme with ¹⁵N-¹³C dipolar mixing times, τ_TEDOR, indicated in the spectra. The fibril samples were prepared from (B) 1,3-¹³C,¹⁵N-huPrP23-144, (C) a mixture of 1,3-¹³C,¹⁵N-huPrP23-144 and natural abundance (¹²C,¹⁴N) huPrP23-144 in a 1:3 molar ratio, and (D) a mixture of 1,3-¹³C-huPrP23-144 and ¹⁵N-huPrP23-144 in a 1:1 molar ratio. The experiment times for spectra in panels (B–D) were ~14, ~27 and ~212 h, respectively. (E–G) Similar to panels (B–D), but for ¹⁵N-¹³Cα (NCA) spectra and fibril samples grown from huPrP23-144 prepared using [2-¹³C] instead of [1,3-¹³C]glycerol. The experiment times were ~7, ~27 and ~216 h, respectively.

Figure 2

Experimental (points) and simulated (lines) 1D TEDOR trajectories that report simultaneously on all intermolecular (A) ¹⁵N-¹³CO and (C) ¹⁵N-¹³Cα distances in huPrP23-144 amyloid fibrils. Experimental trajectories in panels (A) and (C) were recorded for the mixed 1,3-¹³C/¹⁵N and 2-¹³C/¹⁵N fibril samples, respectively, as described in the Supporting Information and the ¹³CO and ¹³Cα intensities in the plots are scaled by corresponding intensities in CP ¹³C reference spectra recorded under identical conditions. The total experiment times were ~41 h and ~68 h, respectively. (B, D) Model spin systems, based on the X-ray structure of amyloid-like microcrystals of the peptide GNNQQNY displaying a parallel in-register alignment of the β-strands (PDB ID 2OMM), used to generate the simulated TEDOR trajectories corresponding to experimental data in panels (A) and (C), respectively, within the SPINEVOLUTION software, with the atoms included in the simulations indicated by spheres. The simulated trajectories were generated as described in detail in the Supporting Information, as a function of a single distance parameter, d, corresponding to the spacing (in Å) between adjacent β-strands as indicated in the structural models. The best-fit ¹⁵N-¹³CO and ¹⁵N-¹³Cα simulations, corresponding to d = 4.4 Å, are shown as solid lines and simulations for d values of ±10% from the best-fit are shown as dashed lines.