Structural properties of Abeta protofibrils stabilized by a small molecule

Abstract

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of Abeta protofibrils. Library screening yielded several molecules that stimulate Abeta aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts Abeta(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37 degrees C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-Abeta aggregates exhibit a low thioflavin T response. Like Abeta fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-Abeta aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous Abeta fibril formation reaction: approximately 12 of the 39 Abeta(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the Abeta molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in Abeta conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester Abeta in a form and/or location that cannot engage the toxic pathway.

Fig. 1.

Profile for screening the LOPAC library of 640 compounds for inhibition of Aβ elongation. (Inset) Structure of CLC.

Fig. 2.

Elongation activity of various Aβ(1-40) aggregates under different conditions. (a) CLC effect on Aβ deposition in microplate assay: CLC present in wells with immobilized fibrils (♦), CLC present in wells with no fibrils (▪), and fibril in wells but no CLC (▴). (b) Dose-response curve for CLC effect on Aβ deposition. (c) Seeding abilities of Aβ aggregates grown in the presence of 100 μMCLC for 2 d (□) and 14 d (▴), or aggregates grown in the absence of CLC for 2 d (•) and 14 d (♦).

Fig. 3.

Formation and characterization of CLC-Aβ(1-40) aggregates. (a) Solution-phase aggregation reaction of Aβ(1-40) monitored by ThT (▴ and ♦) and HPLC of centrifugation supernatants (• and ▪). Disaggregated Aβ(1-40) was incubated alone (▪ and ♦) or with 100 μM CLC (• and ▴). (b) Solution-phase time course of reaction of 100 μM CLC with Aβ(1-40) variants. wt, ⋄; F4P, •; V18P, ▵; I31P, □; F19P/I32P, □. (c) Ab binding to Aβ(1-40) aggregates. WO1 binding to Aβ amyloid fibrils (▪), CLC-Aβ aggregates (•), and a no aggregate control (♦); control IgM Ab binding to Aβ amyloid fibrils (□), CLC-Aβ aggregates (□), and a no aggregate control (⋄).

Fig. 4.

Protofibrillar morphology of CLC-Aβ(1-40) aggregates. Electron micrographs of aliquots of Aβ(1-40) aggregation reactions in the absence [2 d (a) and 14 d (b)] or presence [2 d (c) and 14 d (d)] of 100 μM CLC. Aggregates were adsorbed onto carbon-coated copper grids and negatively stained with either 0.5% uranyl acetate (a Inset and b) or 1% potassium phosphotungstate and photographed on a Hitachi H-800 EM. (a Inset) Adapted from Fig. 1 of ref. .

Fig. 5.

H-bonded structures of various Aβ(1-40) aggregates. HX of various Aβ(1-40) aggregates, corrected for back and forward exchange (17) into side chains only (a) or into side chains plus main chain (b). Fitted curves for repeated runs on monomers (- · · - · · -) and fibrils (―). Averaged data for exchange into protofibrils taken from ref. , ▪; 2-d product of incubation of Aβ(1-40) with 100 μM CLC, ♦.

Fig. 6.

Sensitivity of various sequence positions of Aβ(1-40) to proline replacement in fibrils and protofibrils. Aβ(1-40) concentration in centrifugation supernatants after incubating various mutant peptides with 100 μM CLC for 2 d (a) or with Aβ(1-40) seed fibrils (b) until equilibrium is reached (data from ref. 15). More positive values signify lower aggregate stability. Codes for double proline mutants: P2, 23 and 30; P4, 9, 23, 30, and 37; and β2, 19 and 32.