The absence of high-resolution structures of amyloid oligomers constitutes a major gap in our understanding of amyloid diseases. A growing body of evidence indicates that oligomers of the β-amyloid peptide Aβ are especially important in the progression of Alzheimer’s disease. In many Aβ oligomers, the Aβ monomer components are thought to adopt a β-hairpin conformation. This paper describes the design and study of a macrocyclic β-hairpin peptide derived from Aβ16–36. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and size exclusion chromatography studies show that the Aβ16–36 β-hairpin peptide assembles in solution to form hexamers, trimers, and dimers. X-ray crystallography reveals that the peptide assembles to form a hexamer in the crystal state and that the hexamer is composed of dimers and trimers. Lactate dehydrogenase release assays show that the oligomers formed by the Aβ16–36 β-hairpin peptide are toxic toward neuronally derived SH-SY5Y cells. Replica-exchange molecular dynamics demonstrates that the hexamer can accommodate full-length Aβ. These findings expand our understanding of the structure, solution-phase behavior, and biological activity of Aβ oligomers and may offer insights into the molecular basis of Alzheimer’s disease.

Oligomeric assemblies of the protein α-synuclein are thought to cause neurodegeneration in Parkinson’s disease and related synucleinopathies. Characterization of α-synuclein oligomers at high resolution is an outstanding challenge in the field of structural biology. The absence of high-resolution structures of oligomers formed by α-synuclein impedes understanding the synucleinopathies at the molecular level. This paper reports the X-ray crystallographic structure of oligomers formed by a peptide derived from residues 36–55 of α-synuclein. The peptide 1a adopts a β-hairpin structure, which assembles in a hierarchical fashion. Three β-hairpins assemble to form a triangular trimer. Three copies of the triangular trimer assemble to form a basket-shaped nonamer. Two nonamers pack to form an octadecamer. Molecular modeling suggests that full-length α-synuclein may also be able to assemble in this fashion. Circular dichroism spectroscopy demonstrates that peptide 1a interacts with anionic lipid bilayer membranes, like oligomers of full-length α-synuclein. LDH and MTT assays demonstrate that peptide 1a is toxic toward SH-SY5Y cells. Comparison of peptide 1a to homologues suggests that this toxicity results from nonspecific interactions with the cell membrane. The oligomers formed by peptide 1a are fundamentally different than the proposed models of the fibrils formed by α-synuclein and suggest that α-Syn36–55, rather than the NAC, may nucleate oligomer formation.

Soluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer's disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe, Phe, and Ile were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe side chains and is clasped by the Ile side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.

Amyloid diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes share common features of toxic soluble protein oligomers. There are no structures at atomic resolution of oligomers formed by full-length amyloidogenic peptides and proteins, and only a few structures of oligomers formed by peptide fragments. The paucity of structural information provides a fundamental roadblock to understanding the pathology of amyloid diseases and developing preventions or therapies. Here, we present the X-ray crystallographic structures of three families of oligomers formed by macrocyclic peptides containing a heptapeptide sequence derived from the amyloidogenic E chain of β 2 -microglobulin (β 2 m). Each macrocyclic peptide contains the heptapeptide sequence β 2 m63-69 and a second heptapeptide sequence containing an N-methyl amino acid. These peptides form β-sheets that further associate into hexamers, octamers, and dodecamers: the hexamers are trimers of dimers; the octamers are tetramers of dimers; and the dodecamers contain two trimer subunits surrounded by three pairs of β-sheets. These structures illustrate a common theme in which dimer and trimer subunits further associate to form a hydrophobic core. The seven X-ray crystallographic structures not only illustrate a range of oligomers that a single amyloidogenic peptide sequence can form, but also how mutation can alter the size and topology of the oligomers. A cocrystallization experiment in which a dodecamerforming peptide recruits a hexamer-forming peptide to form mixed dodecamers demonstrates that one species can dictate the oligomerization of another. These findings should also be relevant to the formation of oligomers of full-length peptides and proteins in amyloid diseases.

The assembly of the β-amyloid peptide, Aβ, into soluble oligomers is associated with neurodegeneration in Alzheimer's disease. The Aβ oligomers are thought to be composed of β-hairpins. Here, the effect of shifting the residue pairing of the β-hairpins on the structures of the oligomers that form is explored through X-ray crystallography. Three residue pairings were investigated using constrained macrocyclic β-hairpins in which Aβ30–36 is juxtaposed with Aβ17–23, Aβ16–22, and Aβ15–21. The Aβ16–22–Aβ30–36 pairing forms a compact ball-shaped dodecamer composed of fused triangular trimers. This dodecamer may help explain the structures of the trimers and dodecamers formed by full-length Aβ.