Parallel beta-sheets and polar zippers in amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p

Abstract

We report the results of solid-state nuclear magnetic resonance (NMR) and atomic force microscopy measurements on amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p (Ure2p(10)(-)(39)). Measurements of intermolecular (13)C-(13)C nuclear magnetic dipole-dipole couplings indicate that Ure2p(10)(-)(39) fibrils contain in-register parallel beta-sheets. Measurements of intermolecular (15)N-(13)C dipole-dipole couplings, using a new solid-state NMR technique called DSQ-REDOR, are consistent with hydrogen bonds between side chain amide groups of Gln18 residues. Such side chain hydrogen bonding interactions have been called "polar zippers" by M. F. Perutz and have been proposed to stabilize amyloid fibrils formed by peptides with glutamine- and asparagine-rich sequences, such as Ure2p(10)(-)(39). We propose that polar zipper interactions account for the in-register parallel beta-sheet structure in Ure2p(10)(-)(39) fibrils and that similar peptides will also exhibit parallel beta-sheet structures in amyloid fibrils. We present molecular models for Ure2p(10)(-)(39) fibrils that are consistent with available experimental data. Finally, we show that solid-state (13)C NMR chemical shifts for (13)C-labeled Ure2p(10)(-)(39) fibrils are insensitive to hydration level, indicating that the fibril structure is not affected by the presence or absence of bulk water.

Figure 1

Atomic force microscope image of Ure2p_10–39 fibrils, deposited on mica and recorded in air in tapping mode.

Figure 2

(a) Solid state ¹³C NMR spectra of Ure2p_10–39 in fibrillar and nonfibrillar (crude) form. The peptide is ¹³C-labeled at the carbonyl carbon of Phe37, the α-carbon of Gly22, and the β-carbon of Ala15 (AGF), or ¹³C-labeled at the β-carbon of Gly22 and uniformly ¹⁵N,¹³C-labeled at Gln18 (AQ). (b) Solid state ¹⁵N NMR spectra of Ure2p_10–39-AQ. (c,d) Solid state ¹³C and ¹⁵N NMR spectra of polycrystalline L-glutamine, in which 5% of the molecules are uniformly ¹³C,¹⁵N-labeled. All spectra are recorded with cross-polarization, magic-angle spinning, and proton decoupling. Asterisks indicate spinning sidebands. Signals near 130 ppm in ¹³C NMR spectra are probe background signals.

Figure 3

(a) Measurements of intermolecular ¹³C-¹³C dipole-dipole couplings in Ure2p_10–39-AGF fibrils, using the fpRFDR-CT solid state NMR technique. Each Phe37 carbonyl or Ala15 β-carbon NMR peak is plotted in a 2.0 kHz spectral window. Peaks for dephasing times between 0 and 72 ms are concatenated to show the dipolar dephasing curves. (b) Comparison of experimental and simulated fpRFDR-CT curves, with intermolecular ¹³C-¹³C distances between 4.0 Å and 7.0 Å in the simulations. Error bars represent the root-mean-squared noise in the experimental spectra. Intermolecular distances are determined to be 5.0 ± 0.5 Å for both the Phe37 carbonyl and the Ala15 β-carbon, consistent with an in-register parallel β-sheet structure in the fibrils.

Figure 4

(a) Radio-frequency pulse sequences for measurements of S₁ and S₂ in DSQ-REDOR experiments. The magic-angle spinning period τ_R is 100 μs. XY represents a train of ¹⁵N π pulses with XY-16 phase patterns (76). TPPM represents two-pulse phase modulation(77). In these experiments, M = N₁ = 4, N₂ + N₃ = 48, and N₂ is incremented from 0 to 48 to produce effective dephasing times from 0 to 9.6 ms. Signals arising from intraresidue ¹⁵N-¹³C double single-quantum (DSQ) coherence are selected by phase cycling, with ξ₁ = 0, π, 0, π, 0, π, 0, π, 0, π, 0, π, 0, π, 0, π; ξ₂ = 0, 0, π, π, 0, 0, π, π, 0, 0, π, π, 0, 0, π, π; ξ₃ = 0, 0, 0, 0, π, π, π, π, 0, 0, 0, 0, π, π, π, π; ξ₄ = 0, 0, 0, 0, 0, 0, 0, 0, π, π, π, π, π, π, π, π. Signals are coadded according to the pattern +, −, −, +, +, −, −, +, −, +, +, −, −, +, +, −. (b) Measurements of intermolecular ¹⁵N-¹³C dipole-dipole couplings for glutamine sidechain amide groups in Ure2p_10–39-AQ fibrils (squares) and in polycrystalline 5%-U-¹³C,¹⁵N-Gln (circles), using the DSQ-REDOR solid state NMR technique. To correct for NMR signal decay due to rf pulse imperfections and nuclear spin relaxation, DSQ-REDOR points are obtained by dividing ¹³C NMR signal amplitudes from a constant-time DSQ-REDOR measurement (S₁) by ¹⁵N NMR signal amplitudes (S₂). The vertical scale is therefore in arbitrary units. Solid and dashed lines are least-squares fits to second-order polynomial functions. Error bars represent uncertainties derived from the root-mean-squared noise in the experimental spectra. (c) Experimental S₁ and S₂ spectra from which the DSQ-REDOR data for Ure2p_10–39-AQ fibrils are obtained.

Figure 5

(a) Geometry for isotopically labeled glutamine sidechain amide groups in DSQ-REDOR simulations. Simulations assume a ¹⁵N-¹³C amide bond length d₁ = 1.338 Å, a variable intermolecular distance d₂, and a variable angle θ. (b) Contour plot of the χ² deviation between experimental DSQ-REDOR data for Ure2p_10–39-AQ fibrils and simulated DSQ-REDOR data as a function of the values of d₂ and θ in the simulations. Good fits correspond to χ² ≈ 10 or less. (c) Comparison of experimental and simulated DSQ-REDOR curves for d₂ = 4.80 Å. The angle θ varies from 0° (black line) to 60° (yellow line). (d) Molecular model for glutamine sidechains pendent from an in-register parallel β-sheet in an amyloid fibril, with θ = 25°. Orange atoms are β-carbons of residues that immediately precede and follow the glutamine residue in each β-strand, showing that these β-carbons are on the opposite face of the β-sheet. The spacing between β-strands is 4.80 Å. This geometry permits polar zipper interactions among glutamine sidechains, as proposed by Perutz (,–56). (Model generated in MOLMOL (80).)

Figure 6

(a) ¹³C solid state NMR spectra of Ure2p_10–39-AQ fibrils in a fully hydrated state after fibril formation (wet pellet), after lyophilization, and after rehydration. Minor changes in ¹³C NMR linewidths, but no changes in ¹³C NMR chemical shifts, are observed. Signal-to-noise for the wet pellet is lower and background signal (hump centered at 30 ppm) is higher because of the smaller sample quantity. (b) Proton NMR spectra of the lyophilized and rehydrated samples, indicating the absence of detectable mobile water (sharp peak at 5 ppm) in the lyophilized state. Asterisks indicate spinning sidebands.

Figure 7

Proposal regarding the preference for in-register parallel (left) or antiparallel (right) β-sheets in amyloid fibrils formed by peptides with glutamine- and asparagine-rich sequences. Sequences with asymmetric distributions of glutamine and asparagine residues (such as Ure2p_10–39) can maximize their polar zipper interactions only in an in-register parallel β-sheet, making this the preferred structure. Sequences with palindromic distributions of glutamine and asparagine residues can maximize their polar zipper interactions in either type of β-sheet, allowing electrostatic or other interactions to determine the preferred structure.

Figure 8

Structural models for Ure2p_10–39 fibrils generated by restrained molecular dynamics and energy minimization simulations for a pentameric assembly of Ure2p_10–39 molecules. Both the final energy-minimized structure of the pentamer (right) and the conformation of the central molecule in the pentamer (left) are shown. The two models differ in the relative orientation of the β-sheets formed by the two β-strand segments (residues 10–21 and 27–39), as dictated by the conformation of the intervening loop segment (residues 22–26). (a) Model in which all charged sidechains are outside the fibril core. (b) Model in which oppositely charged sidechains of Arg17 and Asp31 can form internal salt bridges in the fibril core.