3D structure of amyloid protofilaments of beta2-microglobulin fragment probed by solid-state NMR

Abstract

Understanding the structure and formation of amyloid fibrils, the filamentous aggregates of proteins and peptides, is crucial in preventing diseases caused by their deposition and, moreover, for obtaining further insight into the mechanism of protein folding and misfolding. We have combined solid-state NMR, x-ray fiber diffraction, and atomic force microscopy to reveal the 3D structure of amyloid protofilament-like fibrils formed by a 22-residue K3 peptide (Ser(20)-Lys(41)) of beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis. Although a uniformly (13)C,(15)N-labeled sample was used for the NMR measurements, we could obtain the 3D structure of the fibrils on the basis of a large number of structural constraints. The conformation of K3 fibrils was found to be a beta-strand-loop-beta-strand with each K3 molecule stacked in a parallel and staggered manner. It is suggested that the fibrillar conformation is stabilized by intermolecular interactions, rather than by intramolecular hydrophobic packing as seen in globular proteins. Together with thermodynamic studies of the full-length protein, formation of the fibrils is likely to require side chains on the intermolecular surface to pack tightly against those of adjacent monomers. By revealing the structure of beta(2)-microglobulin protofilament-like fibrils, this work represents technical progress in analyzing amyloid fibrils in general through solid-state NMR.

Fig. 1.

AFM images and x-ray fiber diffraction of K3 fibrils. (A) AFM images of K3 fibrils formed in 20% (vol/vol) TFE/10 mM HCl. The scan was performed with a 25-fold diluted sample on a freshly cleaved mica surface. The white scale bar represents 500 nm, and the scan size is 2.5 × 2.5 μm with 512 × 512 points. (B) X-ray fiber diffraction of the K3 fibrils with incident beam perpendicular to the fibril axis. The data shows a typical cross-β pattern. The diffractions corresponding to 4.72 Å (red) and 9.52 Å (blue) indicate the distance between β-strands in the β-sheet and β-sheet layers in the laminated structure, respectively.

Fig. 2.

¹³C–¹³C and ¹⁵N–¹³C correlation NMR spectra of K3 fibrils. (A) 2D broadband ¹³C–¹³C correlation spectra for intraresidue correlations. The figure shows the expansion of the cross-peak regions for aliphatic-carbonyl carbons and aliphatic-aliphatic carbons. This spectrum was obtained at 16.4 T, with a MAS frequency of 16.0 kHz and a 4.00-ms mixing period during which an RFDR pulse sequence was applied. (B) 2D slices of the 3D ¹⁵N–¹³C intraresidue (NCACX) and sequential (NCOCX) correlation spectra. Black and red spectra indicate intraresidue and interresidue correlations, respectively. These spectra were obtained at 11.8 T and a MAS frequency of 12.5 kHz with 1.25-ms and 2.00-ms mixing periods during which the RFDR pulse sequence was applied for NCACX and NCOCX, respectively. Red lines indicate the sequential assignments.

Fig. 3.

2D ¹³C–¹³C spin-diffusion experiments (DARR) with the fully labeled fibrils and spin-diluted fibrils. These spectra were recorded at 16.4 T with a 350-ms mixing period for the spin-diffusion experiment at a 16.0–kHz MAS frequency. (A, C, and E) The ¹³C–¹³C correlation spectrum obtained with the fully labeled fibrils. (B, D, and F) The ¹³C–¹³C correlation spectrum obtained with the spin-diluted fibrils. (C and D) Expansions of aliphatic regions. (E and F) Expansions of the cross-peak regions between aliphatic and aromatic carbons. The arrows indicate the regions of MAS side bands of aromatic carbon signals.

Fig. 4.

2D ¹³C–¹³C correlation spectra obtained with ¹H–¹H spin-diffusion (CHHC) experiments. (A) The spectrum obtained with fully labeled fibrils. The correlation between C^α–C^α carbons is indicated. (B) The spectrum obtained with spin-diluted fibrils. (C) Expansion of the C^α carbon region in the spectrum of fully labeled fibrils. (D) Expansion of the C^α carbon regions in the spectrum of spin-diluted fibrils. Note that the C^α–C^α cross-peaks are significantly reduced. This reduction indicates that the cross-peaks contain intermolecular correlations. These spectra were obtained at 14.1 T with a 210-μs mixing period for the ¹H–¹H spin-diffusion experiment at a MAS frequency of 14.0 kHz.

Fig. 5.

3D structures of tetrameric K3 and monomeric K3 in the fibrillar state. The conformation of K3 in the fibrillar state obtained by simulated annealing molecular dynamics by using CNS. (A) Calculated ensemble of tetrameric structures of K3 fibrils. (B) Ribbon model representation of tetrameric K3 in parallel STAG(+1) conformation. (C) The conformation of one K3 structure in the fibrillar state. (D) Comparison of the conformation of the K3 region in the crystal structure of native β2-m. Notably, the residues between Phe²² and Ser²⁸ are flipped relative to the crystal structure of native β2-m in the fibrillar state.