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. 2009 Nov 10;106(45):18990-5.
doi: 10.1073/pnas.0910080106. Epub 2009 Oct 28.

Molecular basis for insulin fibril assembly

Magdalena I Ivanova  1 , Stuart A Sievers, Michael R Sawaya, Joseph S Wall, David Eisenberg
Affiliations

Molecular basis for insulin fibril assembly

Magdalena I Ivanova et al. Proc Natl Acad Sci U S A. .

Abstract

In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of full-length insulin in a molar ratio-dependent manner, suggesting that this segment is central to the cross-beta spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 A resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated beta-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
An eight-residue segment from the insulin B chain accelerates and inhibits insulin fibril formation. (A) Amino acid sequence of insulin. Segment SLYQLENY of the A chain is dark red. Segment LVEALYLV of the B chain is dark blue. Disulfide bonds are colored in yellow. (B) SLYQLENY of the A chain, when added to the reaction mixture, does not affect the rate of full-length insulin fibril formation. Fibrillation was monitored as a function of time by measuring ThT fluorescence. Full-length insulin starts to form fibrils after 8–10 h, and its conversion is complete in ≈20 h. (C) Fibrillation assay showing LVEALYLV from the B chain inhibits insulin fibril formation at a concentration 10 times less than the concentration of insulin. LVEALYLV also accelerates fibril formation when present at concentrations 25–40 times less than the concentration of insulin. Note that neither SLYQLENY (A chain) nor LVEALYLV (B chain) fluoresce in the presence of ThT. All points represent the mean value of at least four replicates, with error bars representing the SD. (D) Electron micrographs showing that SLYQLENY and LVEALYL aggregates are fibrillar in morphology (E). LVEALYLV in equimolar ratios inhibits insulin fibril formation. Many fibrils were observed in the micrograph of the sample of full-length insulin taken 48 h after the beginning of fibrillation assay (left). In contrast, there were only a few fibrils in the sample of insulin incubated at equimolar ratio with LVEALYLV (right). (Scale bars, 400 nm.)
Fig. 2.
Fig. 2.
LVEALYL from B chain is the smallest segment that can alter the rate of insulin fibril formation. (A) Electron micrograph of fibrillar aggregates of B-chain LVEALYL. (B) Electron micrograph of sample taken from equimolar solution of insulin and LVEALYL after 48 h from beginning of assay. Note that there are only sparse fibril-like aggregates. (C) Fibrillation assay showing that B-chain LVEALYL accelerates insulin fibril formation when added to the reaction mixture at low concentrations, but inhibits insulin fibril formation at higher concentrations. (Scale bars, 400 nm.)
Fig. 3.
Fig. 3.
Atomic structure of B-chain segment LVEALYL, which forms fibril-like microcrystals. (A) View perpendicular to fibril axis showing pair of β-sheets formed by LVEALYL molecules. Crystal needle length runs vertical in this orientation. (B) View down fibril axis showing one layer of interdigitated pair of LVEALYL molecules, which interlock tightly to form the dry steric zipper interface. Pairs of extended β-strands of LVEALYL are stacked in register upon each other, so this figure may be thought of as a projection of two β-sheets, each containing some 100,000 layers. Note that this dry steric zipper interface is devoid of water molecules (shown in yellow). (C) Packing of LVEALYL molecules in crystal, viewed down fibril axis as in (B). Molecules forming the dry steric zipper interface are purple and are separated by water molecules from the next sheets in gray. Thus in LVEALYL crystals one side of each sheet faces water molecules (wet interface).
Fig. 4.
Fig. 4.
STEM measurements of MPL of insulin fibrils. (A) MPL value of the most abundant fibrils was measured as 2.85 ± 0.35 kDa/Å, which is comparable to MPL value of 2.47 kDa/Å of the insulin fibril model shown in Fig. 5. The insulin fibril model contains two molecules of insulin per 4.7-Å layer. MPL values of the thicker fibrils correspond to fibrils with four, six, and eight molecules of insulin per 4.7-Å layer. Histogram was produced by using a binning window of 0.25 kDa/Å. (B) Electron micrographs of insulin fibrils representing the MPL of the fibril populations shown in (A). Note that fibrils with various MPL values display similar morphologies and cross-over distances, suggesting that particles with larger MPL values comprise fibrils with the smallest MPL value of 2.85 kDa/Å. (Scale bars, 100 nm.)
Fig. 5.
Fig. 5.
Fibril model of insulin. (Left) Native structure of the insulin dimer (PDB code 1GUJ). A and B chains of insulin molecule are shown in pale red and pale blue, respectively. The LVEALYL segment, which forms the spine of the fibril, is in dark blue. The SLYQLENY segment, from the A chain, which forms auxiliary sheets to the spine of the fibril, is in dark red. Disulfide bonds are shown in yellow. (Middle) View down fibril axis of four β-sheets of crystal structure of B chain LVEALYL. The two sheets forming the dry steric zipper interface are in blue. Water molecules are shown as green spheres. (Right) View of fibril model, looking down fibril axis. One layer of fibril model is made by stretching both monomers of native insulin (left) in a horizontal direction, converting the deep blue helix of the B chain and the deep red helix of the A chain into extended β-strands. These extended β-strands are given the conformations of the four chain segments of the crystal structure shown in the middle. Thus the spine of the fibril consists of a dry steric zipper formed by the mating of the central two LVEALYL strands from the B chains of the two insulin molecules, plus two outer strands from the A chains of the two molecules.
Fig. 6.
Fig. 6.
Comparison ofobserved and calculated fibril diffraction patterns of insulin fibrils. (A) Cross-β x-ray diffraction pattern of oriented insulin fibrils. On the meridian (vertical axis), there is one strong reflection at 4.7 Å, corresponding to separation of strands within each β-sheet. The weaker reflections on the equator (horizontal axis) are at 9.0 Å and 11.7 Å, arising from separations between β-sheets. (B) Simulated fibril diffraction pattern calculated from the model of the insulin fibril of Fig. 5. An excellent agreement of the diffraction pattern of the model with the observed pattern is noticeable, particularly the 4.7 Å reflection on the meridian and the 9.0 Å and 11.7 Å reflections on the equator.
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References

    1. Westermark P, et al. Amyloid: Toward terminology clarification. Report from the Nomenclature Committee of the International Society of Amyloidosis. Amyloid. 2005;12:1–4. - PubMed
    1. Dische FE, et al. Insulin as an amyloid-fibril protein at sites of repeated insulin injections in a diabetic patient. Diabetologia. 1988;31:158–161. - PubMed
    1. Storkel S, Schneider HM, Muntefering H, Kashiwagi S. Iatrogenic, insulin-dependent, local amyloidosis. Lab Invest. 1983;48:108–111. - PubMed
    1. Brange J, Andersen L, Laursen ED, Meyn G, Rasmussen E. Toward understanding insulin fibrillation. J Pharm Sci. 1997;86:517–525. - PubMed
    1. Westermark P. Aspects on human amyloid forms and their fibril polypeptides. FEBS J. 2005;272:5942–5949. - PubMed

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