SAA fibrils involved in AA amyloidosis are similar in bulk and by single particle reconstitution: A MAS solid-state NMR study

Abstract

AA amyloidosis is one of the most prevalent forms of systemic amyloidosis and affects both humans and other vertebrates. In this study, we compare MAS solid-state NMR data with a recent cryo-EM study of fibrils involving full-length murine SAA1.1. We address the question whether the specific requirements for the reconstitution of an amyloid fibril structure by cryo-EM can potentially yield a bias towards a particular fibril polymorph. We employ fibril seeds extracted from in to vivo material to imprint the fibril structure onto the biochemically produced protein. Sequential assignments yield the secondary structure elements in the fibril state. Long-range DARR and PAR experiments confirm largely the topology observed in the ex-vivo cryo-EM study. We find that the β-sheets identified in the NMR experiments are similar to the β-sheets found in the cryo-EM study, with the exception of amino acids 33-42. These residues cannot be assigned by solid-state NMR, while they adopt a stable β-sheet in the cryo-EM structure. We suggest that the differences between MAS solid-state NMR and cryo-EM data are a consequence of a second conformer involving residues 33-42. Moreover, we were able to characterize the dynamic C-terminal tail of SAA in the fibril state. The C-terminus is flexible, remains detached from the fibrils, and does not affect the SAA fibril structure as confirmed further by molecular dynamics simulations. As the C-terminus can potentially interact with other cellular components, binding to cellular targets can affect its accessibility for protease digestion.

Keywords: Amyloid fibrils; Cryo electron microscopy (EM); Magic Angle Spinning (MAS); Solid-state NMR; Structure.

Graphical abstract

Fig. 1

Biophysical characterization of SAA aggregation. (A) CD spectra of 50 μM SAA recorded as a function of time. At 4 °C, SAA initially adopts an α-helical structure, and converts via a random coil structure into an amyloid fibril. (B) ThT aggregation assay of SAA as a function of protein concentration in the absence of seeds. (C) Tryptophan fluorescence emission spectra for a 10 μM SAA sample incubated at 37 °C as a function of time. (D) Aggregation kinetics obtained from the tryptophan fluorescence emission spectra represented in panel (C). The time scale of aggregation is similar as what is observed in the ThT experiments. (E) ThT aggregation assay for a 50 μM SAA sample in the presence and absence of seeds. (F) TEM image of SAA amyloid fibrils prepared by seeding and used for solid-state NMR experiments. The diameter of selected fibrils (marked in white) is indicated.

Fig. 2

Structural characterization of SAA fibrils using MAS solid-state NMR. (A) 2D-¹³C,¹³C DARR and (B) 2D-¹⁵N,¹³Cα correlation spectra of SAA fibrils with assignments. Chemical shifts are deposited in BMRB under the accession code 51285. SC indicates cross peaks originating from side chain resonances. (C) Secondary chemical shifts and β-sheet propensity for SAA fibrils obtained from solid-state NMR. (D) 2D strip plots showing sequential connectivities for residues M16-A19 in SAA fibrils from 3D NCACX, NCOCX, and CONCA experiments (represented in red, green and magenta, respectively). (E, F, G) Proton detected ¹H,¹³C INEPT correlation spectrum, carbon detected ¹H,¹³C INEPT correlation spectrum, and proton detected ¹H,¹⁵N INEPT correlation spectrum, respectively, recorded for a SAA fibril sample in a MAS solid-state NMR rotor. Only dynamic residues are observable in these experiments. Tentative assignments are indicated with dashed circles. Average random coil chemical shifts for each amino acid type are obtained from the BMRB (https://bmrb.io/). (H) Amino acid types that are observed in the INEPT spectra are highlighted with arrows.

Fig. 2

Structural characterization of SAA fibrils using MAS solid-state NMR. (A) 2D-¹³C,¹³C DARR and (B) 2D-¹⁵N,¹³Cα correlation spectra of SAA fibrils with assignments. Chemical shifts are deposited in BMRB under the accession code 51285. SC indicates cross peaks originating from side chain resonances. (C) Secondary chemical shifts and β-sheet propensity for SAA fibrils obtained from solid-state NMR. (D) 2D strip plots showing sequential connectivities for residues M16-A19 in SAA fibrils from 3D NCACX, NCOCX, and CONCA experiments (represented in red, green and magenta, respectively). (E, F, G) Proton detected ¹H,¹³C INEPT correlation spectrum, carbon detected ¹H,¹³C INEPT correlation spectrum, and proton detected ¹H,¹⁵N INEPT correlation spectrum, respectively, recorded for a SAA fibril sample in a MAS solid-state NMR rotor. Only dynamic residues are observable in these experiments. Tentative assignments are indicated with dashed circles. Average random coil chemical shifts for each amino acid type are obtained from the BMRB (https://bmrb.io/). (H) Amino acid types that are observed in the INEPT spectra are highlighted with arrows.

Fig. 3

Comparison of the SAA fibril topology from solid-state NMR and cryo-EM. (A) Secondary structure elements of SAA fibrils from solid-state NMR and cryo-EM (Liberta et al., 2019a, Bansal et al., 2021). (B) Long-range distance contacts in SAA fibrils were observed in solid-state NMR experiments. The small panels highlight the cross-peaks involving Y20-W28, I6-V51, F10-W17, and R18-D30, respectively. (C) Cryo-EM ex-vivo fibril structure (PDB ID: 6dso). Residues that could be sequentially assigned by MAS solid-state NMR are highlighted in grey. Long-range distance restraints from NMR experiments are indicated by dashed lines.

Fig. 4

Structure and conformational dynamics from MD simulations. (A) Simulation snapshots of the four fibril structures after 100 ns: 6dso, 6zch, model 1 and model 2. Residues at the protofilament interface are shown in licorice representation. Interfaces involve the following amino acids; 6dso: D59, R61; 6zch: G1, D22, K24, E25; model 1: E25, K29; model 2: G1, D22, K24, E25, E55, K56, D59, R61. (B) Superposition of one central chain of simulated structures after 100 ns and the experimental cryo-EM structure 6dso (shown in green). (C) RMSF averaged over each residue for the experimental cryo-EM structures 6dso and 6zch (left) and the two models with alternative interface (right). Error bars correspond to standard deviations. Dashed vertical lines indicate the residues at the interface.

Fig. 5

Aromatic region of the 2D ¹³C,¹³C (DARR) correlation spectrum obtained for SAA fibrils. The spectral region containing (A) aliphatic–aromatic and (B) aromatic-aromatic cross-peaks. Distinct resonances for Y20-Cε1 and Cε2 chemical shifts suggest that the aromatic ring of Y20 is rigid. At the same time, the aromatic spin systems of W17, W28, and W52 are well resolved, while H36 and H72/H84 cannot be observed.

Fig. 6

Superposition of solid-state NMR spectra obtained for seeded (black) and non-seeded SAA fibril (blue) preparations. (A) 2D ¹³C,¹³C DARR and (B) 2D ¹⁵N, ¹³Cα correlation spectra. Tentatively assigned cross-peaks of the non-seeded sample are labeled in red. (C) TEM images of non-seeded fibrils. We observe two classes of fibrils. Fibrils consisting of three protofilaments yield a diameter of ca. 18–19 nm. Thinner fibrils that contain two protofilaments have a diameter of ca. 12–13 nm.