Fibril structure of human islet amyloid polypeptide

Abstract

Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two β-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing β-sheets that twist around each other. The two β-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (∼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding.

FIGURE 1.

Electron microscope images of hIAPP fibrils showing twisted morphology. A, negatively stained wild-type hIAPP fibrils. B, unilateral metal shadowing of wild-type hIAPP fibrils showing a left-handed twist (metal was deposited from the right; deposited metal is shown in white, and shadows are black). C, negatively stained singly labeled (13R1) hIAPP fibrils. D, negatively stained double-labeled (13R1/18R1) hIAPP fibrils. Bar: 200 nm.

FIGURE 2.

Residue mobilities of hIAPP fibrils containing 25% R1, as calculated from the inverse of central line width (ΔH_o⁻¹) of EPR spectra shown in supplemental Fig. S2. No mobility data were obtained for residue 13, but residue 12 was included in the strand region based upon the results from the four-pulse DEER distance measurements. The double-headed arrows indicate regions proposed to be in a β-sheet conformation based on solid-state NMR (19). The top panel shows the amino acid sequence of hIAPP 1–37. The boxed areas correspond to the residues outlined in the mobility graph below.

FIGURE 3.

Schematic model of hIAPP in protofilament and four-pulse DEER distance measurements. A, hIAPP with two β-strands regions connected by a loop. Placement of “inward” and “outward” oriented side chains is based on mobility measurements by continuous wave EPR (supplemental Fig. S3). Intrastrand (green) and interstrand (red) distances are indicated schematically. B, intramolecular DEER distances measured in doubly labeled hIAPP peptides within fibrils. Distances are from Gaussian fits of dipolar evolution data (supplemental Fig. S4).

FIGURE 4.

Model of hIAPP fibril structure. A, a typical hIAPP peptide incorporated in the fibril, viewed along the fibril axis. B, the same peptide viewed along an axis orthogonal to the fibril axis, showing the stagger of the two β-strands of the peptide. C, a section of the structural model showing the stagger of the peptides (shown as blue, green, and orange ribbons; the relationship of blue-colored peptides is indicated). D, rotation of the structural model with the axes of β-strands orthogonal to the fibril. E, a structural model containing 101 peptides showing a turn of the left-handed helix of ∼90° between the centers of the red boxes. The helix has an average pitch of 440 Å.