Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the β-Sheet Core

Abstract

Amyloid deposits formed from islet amyloid polypeptide (IAPP) are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic β-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form. Complete sequential resonance assignments and resulting constraints on secondary structure are shown. A single set of chemical shifts is found for most residues, which is indicative of a high degree of homogeneity. The core region comprises three to four β-sheets. We find that the central 23-FGAILS-28 segment, which is of critical importance for amyloid formation, is part of the core region and forms a β-strand in our sample preparation. The eight N-terminal amino acid residues of IAPP, forming a ring-like structure due to a disulfide bridge between residues C2 and C7, appear to be well defined but with an increased degree of flexibility. This study supports the elucidation of the structural basis of IAPP amyloid formation and highlights the extent of amyloid fibril polymorphism.

Fig 1. Atomic force micrographs of IAPP after fibrillation.

Shown are scans of two different areas of the sample (A, B). C and D are additional scans focusing on smaller regions of A and B. Each scan consists of 1024x1024 pixels. Fibrils appear to laterally assemble into bundles.

Fig 2. Proton-Driven-Spin-Diffusion (PDSD) spectrum.

The spectrum was recorded at a field of 18.8 Tesla with longitudinal mixing time of 20 ms and spinning speed of 11 kHz. Sequential assignments shown are based on a number of experiments and brought together in this figure. There are two cross-sections drawn underneath, taken at the positions indicated by dotted lines. The dashed pointers mark the additional peaks found for I26 (Cδ₁) and S29 (Cβ).

Fig 3. Sequential walk in the N-terminus via backbone nitrogen chemical shifts.

The orange and blue peaks belong to a 3D NCACB spectrum (positive, negative) acquired at 14 kHz MAS at 14.1 Tesla. Red peaks belong to a NCACX 3D spectrum recorded at 14.1 Tesla and 11 kHz MAS spinning. Green peaks come from a 3D NCOCX experiment acquired at the same spectrometer at 14 kHz MAS spinning. All experiments were performed at a nominal sample temperature of 10°C. Reading from up to down, a spin system i is assigned to its backbone nitrogen shift in the upper strip. In the strip underneath, the preceding residue (green peaks) i-1 appears at the same nitrogen shift as found above. Deviations of up to 0.7 ppm appear due to the line-widths of the nitrogen chemical shifts.

Fig 4. Analytical RP-HPLC before fibrillation.

Performed under reducing (dashed line) and oxidizing (solid line) conditions to verify the presence of the disulfide bridge between residues C2 and C7.

Fig 5. The effect of low temperature and hyperpolarization on the sample.

Overlay of 2D Single-quantum double-quantum spectra recorded at magnetic fields of 14.1 T. The green spectrum was acquired with Dynamic-Nuclear Polarization (DNP) at 100 Kelvin nominal sample temperature and SPC5_2 recoupling at 8 kHz MAS. The purple spectrum was acquired with conventional solid-state NMR technique at 0°C nominal sample temperature with SPC5_3 recoupling at 11 kHz MAS. In the DNP experiment, the sample was frozen out and it is obvious, that the N-terminal Cα-Cβ cross-peak are broadened due to impeded molecular motion.

Fig 6. The location of β-strands in fibrillar IAPP.

Top, calculated differences of secondary chemical shifts Δδ C_sec = Δδ C_α- Δδ C_β, with Δδ C_x = δ C_x(exp) - δ C_x(BMRB). Two or more adjacent negative values usually are indicative of a β-strand. Bottom, red arrows represent the β-strands predicted by TALOS-N based on NMR chemical shifts from the current study (except for residues A5, T6 and C7, which are part of the disulfide bridged N-terminal loop). Position S29 is found to be structurally less constrained and able to form part of a β-strand in a fraction of fibrils. Grey bars indicate the location of β-strands as determined in previous studies.