Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR

Abstract

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range (13)C-(13)C correlation spectra and the third based on the identification of intermolecular interactions in (13)C-(15)N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient (13)C-(13)C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The (13)C-(13)C experiments are corroborated by (15)N-(13)C correlation spectra obtained from a mixed [(15)N,(12)C]/[(14)N,(13)C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular (15)N-(13)C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular (13)C-(13)C and (15)N-(13)C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.

Figure 1

(a) Sub-section of a BASE RFDR spectrum of microcrystalline 2-G_B1 showing cross-peaks between Y45Cα and neighboring nuclei. (b) Inter-nuclear distances in the crystal structure of G_B1 (PDB ID 2QMT) corresponding to the cross-peaks observed between Y45Cα and other ¹³Cα sites, i.e., within its own strand (T44, D46, and D47), to a strand within the same molecule (T51 and F52), and to an adjacent strand in a neighboring molecule (K13* and G14*). Asterisks denote residues in an adjacent protein molecule in the crystal lattice. The spectrum in (a) was recorded with τ_mix = 24 ms and a total experimental time of 7.5 hours.

Figure 2

Inter-nuclear distances anticipated in parallel β-strands and resolvable ¹³Cα-¹³Cα correlations for a given residue in the middle of three different strands h, i, k (left), and three identical in-register strands i, i, i (right). Inter-strand correlations in the parallel in-register case are degenerate with sequential correlations within the strand. Typical internuclear distances are indicated on the left. Dashed lines of different colors (except for black) indicate the potentially resolved cross-peaks in ¹³C-¹³C correlation spectra.

Figure 3

Section of a BASE RFDR spectrum of amyloid fibrils formed by 2-PI3-SH3. Gray labels indicate sequential ¹³Cα-¹³Cα cross-peaks while black labels denote cross-peaks between ¹³Cα nuclei separated by two residues, with an inter-nuclear distance corresponding to ∼6.5 Å. Backbone-backbone correlations between sites distant in space, but near in sequence, are readily observed for several regions of the polypeptide chain. This spectrum was recorded with τ_mix = 24 ms and a total experimental time of 5 days.

Figure 4

Sections of PDSD ¹³C-¹³C correlation spectra acquired with a mixing time of 20 ms optimized for one-bond correlations of (a) U-PI3-SH3 and (b) 2-PI3-SH3, and with a mixing time of 500 ms optimized for long-range correlations in (c) 2-PI3-SH3. The dotted boxes in (a) and (b) correspond to the same region as that shown in (c), in which asterisks identify correlations between neighboring molecules in a parallel, in-register architecture.

Figure 5

(a-c) 750 MHz intermolecular ¹⁵N-¹³C correlations in PI3-SH3 fibrils recorded at 300 K with 16 days of acquisition. The three panels correspond to the ¹⁵N-¹³C=O, aromatic, and ¹⁵N-13Ca regions of the spectra. (d-f) The identical spectral regions recorded at 100 K and 400 MHz with DNP enhancement in 32 hours of signal averaging. The spectra were obtained with ZF-TEDOR recoupling (τ_mix = 16 ms) from a mixed PI3-SH3, a sample fibrillized from a mixture of [¹⁵N] monomers and [2-¹³C] monomers. (g) Illustration of the 23 interstrand contacts established from ¹³C-¹⁵N cross peaks in the 750 MHz spectra acquired at 300 K in (a-c); (h) the 52 interstrand contacts established from the 400 MHz DNP enhanced spectra recorded at 100 K shown in (d-f).

Figure 6

(a) Summary of intermolecular constraints along the PI3-SH3 sequence obtained with the methods described in the text: Indirect CC (“>”), direct CC (“*”), mixed NC at room temperature (“– “), and mixed NC at 100 K with DNP (“+”). Filled bars indicate residues in a β-strand conformation while empty bars mark dynamic regions that have not been assigned in the spectra. (b) Superposition of all intermolecular constraints on a hypothetical model of PI3-SH3 amyloid fibril architecture in which two β-sheet layers (light gray and dark gray, respectively) are formed by each half of the sequence.