Atomic structure of the cross-beta spine of islet amyloid polypeptide (amylin)

Abstract

Human islet amyloid polypeptide (IAPP or amylin) is a 37-residue hormone found as fibrillar deposits in pancreatic extracts of nearly all type II diabetics. Although the cellular toxicity of IAPP has been established, the structure of the fibrillar form found in these deposits is unknown. Here we have crystallized two segments from IAPP, which themselves form amyloid-like fibrils. The atomic structures of these two segments, NNFGAIL and SSTNVG, were determined, and form the basis of a model for the most commonly observed, full-length IAPP polymorph.

Figure 1.

IAPP residues 23–37 are necessary for fibril formation of IAPP. (A) The MBP–IAPP construct forms amyloid fibrils as visualized by electron microscopy. The scale bar represents 100 nm. These fibrils were determined to contain the full-length construct by mass spectrometry. (B) The MBP–IAPP 1–22 construct failed to form fibrils. (C) The fibril-forming sequence of human IAPP is compared to that of the nonfibril-forming mouse IAPP, with residue differences noted in red. (D) The effects of residue replacements on the rate of fibrillation of IAPP. Histidine 18 of human IAPP is the only residue difference between human and mouse IAPP in the N-terminal region. This residue difference appears to have no effect on the fibril formation of IAPP, whereas the subtle F23L replacement delays fibrillation. The double mutant (H18R/F23L) shows an even greater lag time, suggesting cooperativity between these sites during fibril formation. The H18R/S28P/S29P triple mutant shows that although IAPP 22–27 may be required for fibril formation, the downstream proline substitutions can prevent conversion to amyloid fibrils. (E) Insulin increases the lag time prior to fibrillation. This affect appears to be slightly less potent with the H18R substitution.

Figure 2.

Structures of the NNFGAIL and SSTNVG amyloid-like segments of IAPP. The NNFGAIL (A) and SSTNVG (B) segments, which abut in IAPP, are capable of forming amyloid-like fibrils (left panels) and microcrystals (right panels), respectively. The scale bars are 100 nm for the electron microscopy images (left panels) and 50 μm for the light microscopy images (right panels). (C) The structure of NNFGAIL, consisting of two close-packed β-sheets, viewed down the sheets. Notice the pronounced bend in the backbone, but not the usual interpenetration of side chains found in the steric zipper. Instead, NNFGAIL displays a dry, main chain–main chain zipper-like interface. Water molecules are shown as yellow spheres, and are outside the intersheet interface in both NNFGAIL and SSTNVG. (D) The structure of SSTNVG, viewed down the β-sheets shows the interdigitated side chains between adjacent β-sheets across a dry, steric zipper interface. (E) Two layers of the NNFGAIL structure, shown as stick molecules, showing the hydrogen bonds between layers. (F) Five layers of the SSTNVG structure viewed from the wet interface. Notice that E and F are 90° from the views of C and D.

Figure 3.

Model of an IAPP fibril based on our crystal structures of NNFGAIL and SSTNVG. (A) View down the fibril axis (shown by the black dot). Two molecules of IAPP mate in a dry steric zipper around the fibril axis. The segment NNFGAIL, shown in blue in each IAPP molecule, is part of the hairpin turn and the start of the long steric zipper interface. The extension of the central strand, and its mating strand, contain the SSTNVG zipper interface, shown in green in each IAPP molecule. The final four residues of IAPP are modeled to complete the zipper interface. The initial 20 residues of IAPP are modeled to complete the outer strands of the two sheets, and to form the cyclic disulfide bridge near the N terminus. (B) Space-filling representation shows the tight steric zipper interface between residues 23 and 37 on the two IAPP molecules. (C) View down the axis of the fibril with a diameter of 64 Å. The modeled residues are shown in white and the residues corresponding to our crystal structures are shown in blue for NNFGAIL and green for SSTNVG. (D) View perpendicular to the fibril axis of the same fibril with a length of 125 Å (one-quarter of a full turn), showing the 4.8 Å spacing that gives rise to the strong meridional reflection of the fibril diffraction pattern. This model has a mass per unit length and crossover distance that agrees closely with the experimental value for the most commonly observed polymorphic fibril.

Figure 4.

Comparison of the simulated fibril diffraction patterns of fibril models with the experimental diffraction images of IAPP 23–37. The 23–37 segment represents the steric zipper spine of the fibril and is the segment of highest confidence in the model. Space-filling representations are shown for our models (A,C). The simulated fiber diffraction patterns, obtained by Fourier-transforming these models, are shown on the left halves and the experimental diffraction pattern for the IAPP 23–37 segment are shown on the right halves of B and D. The hallmark 4.78 Å meridional reflection characteristic of the hydrogen bonding axis is observed. The arrows in B indicate equatorial reflections at ∼15 Å (red) and ∼8.5 Å (white) that match between the simulated model and experimental diffraction patterns.