The Structure of Human IAPP Fibrils Reflects Membrane and pH Conditions

Abstract

Physiologically relevant in vitro models of amyloid aggregation are essential for linking structural insights to disease pathology. In type 2 diabetes, aggregation of human islet amyloid polypeptide (hIAPP) into fibrils is a hallmark of β-cell dysfunction, yet structural data on ex vivo hIAPP fibrils remain unavailable. Most models use solution-grown fibrils, overlooking membrane interactions and native pH, which underscores the need for more realistic in vitro models. Here, we use solid-state NMR spectroscopy to determine the structure of phospholipid membrane-mediated hIAPP fibrils formed under extracellular (pH 7.4) conditions. These fibrils are homogeneous and adopt an L-shaped protofilament architecture with an extended N-terminal β-strand─a region often unresolved in cryo-EM. The fibril core (N14-L27) adopts the CF1 fold, a conserved β-arch also seen in nonlipidic fibrils, suggesting its relevance in disease. In contrast, fibrils formed at intracellular pH (5.3) are structurally heterogeneous and show distinct structural differences in the C-terminus. hIAPP must exhibit substantial structural plasticity in the membrane environment, transitioning from helical monomers to β-hairpin oligomers and ultimately to β-arch-rich fibrils─transitions that may introduce energy barriers stabilizing toxic intermediates. Our findings provide the first high-resolution structure of membrane-mediated hIAPP fibrils highlighting the need to model aggregation under physiologically relevant conditions.

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Clumped lipidic fibrils: Negative-stain electron micrograph of fibrils formed by 20 μM hIAPP aggregated in the presence of 100 μM POPC:POPS (8:2) vesicles in 10 mM phosphate buffer (pH 7.4) containing 50 mM NaCl, incubated for 66 h at 37 °C.

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Membrane-mediated aggregation kinetics: (A) Time-dependent decay of normalized NMR peak intensity of soluble hIAPP monomers at different lipid-to-peptide ratios (LPRs) and buffer conditions (see legend). All curves except LPR 0 (pH 5.3, no NaCl) were fitted to a sigmoidal function to extract T ₅₀ values, shown as a bar plot with fitting errors (inset). The LPR 0 (pH 5.3) data set was excluded due to negligible decay. For LPR 5 (pH 5.3, no NaCl), a mixed exponential–sigmoidal function was used to capture the initial rapid decay. (B) NMR-derived monomer consumption curves (blue) and normalized ThT fluorescence intensities (red) for various LPRs (see legend). Both data sets were fitted to sigmoidal functions to extract T ₅₀ values (bar plot with fitting errors in inset). The nucleation phase (NP, ThT I/I ₀ < 5) is shaded in red. A 12-h time scale comparison of panels A and B is provided in Figure S4. (C) Normalized NMR peak intensity of the lipid choline signal (3.25 ppm) over time, fitted to a polynomial function, for each LPR and buffer condition. Inset: lipid peak decay during the measurement dead time (T _D, 5 min), and change in choline signal upon addition of 20 μM hIAPP.

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Secondary structure comparison: (A) 2D hCC SPEPS spectra of lipidic hIAPP fibrils grown at pH 7.4 (blue) and pH 5.3 (orange), showing C–C cross-peaks in the aliphatic region. Residues exhibiting significant peak shifts at pH 5.3 are underscored. Alternative peak sets are marked with a prime symbol. (B) TALOS-N predicted φ (open circles, top panel) and ψ (filled circles, middle panel) dihedral angles (360° scale) and associated errors for pH 7.4 lipidic fibrils, compared to residues 14–36 in various S-type cryo-EM structures (see legend). Cryo-EM angle distributions are shown as shaded bars representing the error range around the average angle from four structures per residue. The bottom panel shows root-mean-square deviation (RMSD) of torsional angles between pH 7.4 fibrils and the averaged cryo-EM S-type structures. Residues with low-quality predictions are excluded and marked with asterisks. An enlarged version of panel B is available as Figure S11. (C) TALOS-N prediction confidence for secondary structure elements in pH 7.4 lipidic fibrils. (D) Comparison of β-strand locations (solid arrows) across lipidic fibrils (this study), nonlipidic S-type fibrils (ssNMR and cryo-EM), lipid-bound oligomers in nanodiscs, and monomeric states in SDS micelles. Antiparallel β-strands in oligomers are shown with half a rrows; helices in monomers are represented by blue cylinders. (E) Average chemical shift perturbations (CSPs) between pH 7.4 and pH 5.3 lipidic fibrils. Filled black circles indicate average CSPs for alternative chemical shift sets observed in specific residues at pH 5.3.

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Structure of pH 7.4 lipidic fibrils: (A) Surface-filled structure of a single protofilament representing one molecular layer from the lowest-energy conformer of pH 7.4 lipidic fibrils, built using CYANA. Color-coded by atomic partial charge (blue: positive, red: negative). Inset: stack of four monomers displaying a cross-β structure. Dotted double arrows indicate potential flexibility arising from limited long-range restraints in the flanking N-terminal (residues 1–12) and C-terminal (residues 31–37) regions. (B, C) Ten lowest-energy conformers generated from CYANA calculations, each consisting of four protofilaments arranged in a cross-β architecture. Individual monomers are shown in different colors for clarity. Most conformers exhibit a subtle β-sheet twist (B), while two display a flatter β-sheet arrangement (C). (D) Alignment of a lipidic fibril layer with a nonlipidic S-type fibril composed of two protofilaments in a C2-symmetric arrangement. (E) Superposition of the lipidic protofilament with eight nonlipidic fibrils, aligned on the conserved CF1 fold (residues 14–27); side chains shown as sticks. (F) TW1 polymorph (PDB 7M61) featuring a CF1-type protofilament (aligned with the lipidic protofilament) interfacing with a CF2 protofilament. Molecular layers from (G) early stage and (H) mature S20G hIAPP fibrils. PDB accession codes are color-coded to match the corresponding protofilaments.

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Potential structural reorganization. (A) hIAPP monomers containing helical segments (green cylinders) in SDS micelles (Nanga et al.). (B) Oligomeric β-hairpin motifs with intramolecular hydrogen bonds formed by antiparallel β-strands (green arrows), observed by Rodrigues et al. in lipid nanodiscs. (C) Lipidic fibrils at pH 7.4 containing β-arch motifs formed by parallel-in-register β-strands. Intra- and intermolecular hydrogen bonds in β-hairpin and β-arch structures are shown as pink dotted lines. If (A) and (B) represent intermediates on pathway to lipidic fibrils in (C), structural reorganization such as a hairpin to arch transition may play a critical role in fibril elongation and the stabilization of intermediate states.