Elucidating the Structures of Amyloid Oligomers with Macrocyclic β‑Hairpin Peptides: Insights into Alzheimer’s Disease and Other Amyloid Diseases

ConspectusIn the more than a century since its identification, Alzheimer’s disease has become the archetype of amyloid diseases. The first glimpses of the chemical basis of Alzheimer’s disease began with the identification of “amyloid” plaques in the brain in 1892 and extended to the identification of proteinaceous fibrils with “cross-β” structure in 1968. Further efforts led to the discovery of the β-amyloid peptide, Aβ, as a 40- or 42-amino acid peptide that is responsible for the plaques and fibrils. At this point, a three-decade-long marathon began to elucidate the structure of the fibrils and identify the molecular basis of Alzheimer’s disease. Along the way, an alternative model began to emerge in which small aggregates of Aβ, called “oligomers”, rather than fibrils, are the culprits that lead to neurodegeneration in Alzheimer’s disease. This Account describes what is known about the structures of the fibrils and details our research group’s efforts to understand the structural, biophysical, and biological properties of the oligomers in amyloid diseases.β-Sheets are the building blocks of amyloid fibrils and oligomers. Amyloid fibrils generally consist of extended networks of parallel β-sheets. Amyloid oligomers appear to be more compact enclosed structures, some of which are thought to be composed of antiparallel β-sheets comprising β-hairpins. β-Hairpins are special because their twisted shape, hydrophobic surfaces, and exposed hydrogen-bonding edges impart a unique propensity to form compact assemblies. Our laboratory has developed macrocyclic β-sheets that are designed to mimic β-hairpins formed by amyloidogenic peptides and proteins. The β-hairpin mimics contain two β-strand peptide fragments linked together at their N- and C-termini by two δ-linked ornithine turn mimics to create a macrocycle. An <i>N</i>-methyl group is installed on one of the β-strands to prevent uncontrolled aggregation. These design features facilitate crystallization of the β-hairpin mimics and determination of the X-ray crystallographic structures of the oligomers that they form.During the past few years, our laboratory has elucidated the X-ray crystallographic structures of oligomers formed by β-hairpin mimics derived from Aβ, α-synuclein, and β₂-microglobulin. Out of these three amyloidogenic peptides and proteins, the Aβ β-hairpin mimics have provided the most insight into amyloid oligomers. Our studies have revealed a previously undiscovered mode of self-assembly, whereby three Aβ β-hairpin mimics assemble to form a triangular trimer. The triangular trimers are remarkable, because they contain two largely hydrophobic surfaces that pack together with other triangular trimers to form higher-order oligomers, such as hexamers and dodecamers. Some of the dodecamers pack in the crystal lattice to form annular porelike assemblies. Some of the β-hairpin mimics and triangular trimers assemble in solution to form oligomers that recapitulate the crystallographically observed oligomers. These oligomers exhibit toxicity toward neuronally derived cells, recapitulating the toxicity of the oligomers formed by full-length amyloidogenic peptides and proteins. These findings are significant, because they address a gap in understanding the molecular basis of amyloid diseases. We anticipate that these studies will pave the way for developing diagnostics and therapeutics to combat Alzheimer’s disease, Parkinson’s disease, and other amyloid diseases

Coassembly of Peptides Derived from β‑Sheet Regions of β‑Amyloid

In this paper, we investigate the coassembly of peptides derived from the central and C-terminal regions of the β-amyloid peptide (Aβ). In the preceding paper, <i>J. Am. Chem. Soc.</i> <b>2016</b>, DOI: 10.1021/jacs.6b06000, we established that peptides containing residues 17–23 (LVFFAED) from the central region of Aβ and residues 30–36 (AIIGLMV) from the C-terminal region of Aβ assemble to form homotetramers consisting of two hydrogen-bonded dimers. Here, we mix these tetramer-forming peptides and determine how they coassemble. Incorporation of a single ¹⁵N isotopic label into each peptide provides a spectroscopic probe with which to elucidate the coassembly of the peptides by ¹H,¹⁵N HSQC. Job’s method of continuous variation and nonlinear least-squares fitting reveal that the peptides form a mixture of heterotetramers in 3:1, 2:2, and 1:3 stoichiometries, in addition to the homotetramers. These studies also establish the relative stability of each tetramer and show that the 2:2 heterotetramer predominates. ¹⁵N-Edited NOESY shows the 2:2 heterotetramer comprises two different homodimers, rather than two heterodimers. The peptides within the heterotetramer segregate in forming the homodimer subunits, but the two homodimers coassemble in forming the heterotetramer. These studies show that the central and C-terminal regions of Aβ can preferentially segregate within β-sheets and that the resulting segregated β-sheets can further coassemble

X‑ray Crystallographic Structures of Trimers and Higher-Order Oligomeric Assemblies of a Peptide Derived from Aβ_17–36

A peptide derived from Aβ_17–36 crystallizes to form trimers that further associate to form higher-order oligomers. The trimers consist of three highly twisted β-hairpins in a triangular arrangement. Two trimers associate face-to-face in the crystal lattice to form a hexamer; four trimers in a tetrahedral arrangement about a central cavity form a dodecamer. These structures provide a working model for the structures of oligomers associated with neurodegeneration in Alzheimer’s disease

Elucidation of the Teixobactin Pharmacophore

This paper elucidates the teixobactin pharmacophore by comparing the arginine analogue of teixobactin Arg₁₀-teixobactin to seven homologues with varying structure and stereochemistry. The roles of the guanidinium group at position 10, the stereochemistry of the macrolactone ring, and the “tail” comprising residues 1–5 are investigated. The guanidinium group is not necessary for activity; Lys₁₀-teixobactin is more active than Arg₁₀-teixobactin against Gram-positive bacteria in minimum inhibitory concentration (MIC) assays. The relative stereochemistry of the macrolactone ring is important. Diastereomer l-Thr₈,Arg₁₀-teixobactin is inactive, and diastereomer d-<i>allo</i>-Ile₁₁,Arg₁₀-teixobactin is less active. The macrolactone ring is critical; <i>seco</i>-Arg₁₀-teixobactin is inactive. The absolute stereochemistry is not important; the enantiomer <i>ent</i>-Arg₁₀-teixobactin is comparable in activity. The hydrophobic <i>N</i>-terminal tail is important. Truncation of residues 1–5 results in loss of activity, and replacement of residues 1–5 with a dodecanoyl group partially restores activity. These findings pave the way for developing simpler homologues of teixobactin with enhanced pharmacological properties

Assembly of Peptides Derived from β‑Sheet Regions of β‑Amyloid

In Alzheimer’s disease, aggregation of the β-amyloid peptide (Aβ) results in the formation of oligomers and fibrils that are associated with neurodegeneration. Aggregation of Aβ occurs through interactions between different regions of the peptide. This paper and the accompanying paper constitute a two-part investigation of two key regions of Aβ: the central region and the C-terminal region. These two regions promote aggregation and adopt β-sheet structure in the fibrils, and may also do so in the oligomers. In this paper, we study the assembly of macrocyclic β-sheet peptides that contain residues 17–23 (LVFFAED) from the central region and residues 30–36 (AIIGLMV) from the C-terminal region. These peptides assemble to form tetramers. Each tetramer consists of two hydrogen-bonded dimers that pack through hydrophobic interactions in a sandwich-like fashion. Incorporation of a single ¹⁵N isotopic label into each peptide provides a spectroscopic probe with which to elucidate the β-sheet assembly and interaction: ¹H,¹⁵N HSQC studies facilitate the identification of the monomers and tetramers; ¹⁵N-edited NOESY studies corroborate the pairing of the dimers within the tetramers. In the following paper, <i>J. Am. Chem. Soc.</i> <b>2016</b>, DOI: 10.1021/jacs.6b06001, we will extend these studies to elucidate the coassembly of the peptides to form heterotetramers

X‑ray Crystallographic Structures of Trimers and Higher-Order Oligomeric Assemblies of a Peptide Derived from Aβ_17–36

A peptide derived from Aβ_17–36 crystallizes to form trimers that further associate to form higher-order oligomers. The trimers consist of three highly twisted β-hairpins in a triangular arrangement. Two trimers associate face-to-face in the crystal lattice to form a hexamer; four trimers in a tetrahedral arrangement about a central cavity form a dodecamer. These structures provide a working model for the structures of oligomers associated with neurodegeneration in Alzheimer’s disease

X‑ray Crystallographic Structures of Trimers and Higher-Order Oligomeric Assemblies of a Peptide Derived from Aβ_17–36

A peptide derived from Aβ_17–36 crystallizes to form trimers that further associate to form higher-order oligomers. The trimers consist of three highly twisted β-hairpins in a triangular arrangement. Two trimers associate face-to-face in the crystal lattice to form a hexamer; four trimers in a tetrahedral arrangement about a central cavity form a dodecamer. These structures provide a working model for the structures of oligomers associated with neurodegeneration in Alzheimer’s disease

Polymorphism of Oligomers of a Peptide from β‑Amyloid

This contribution reports solution-phase structural studies of oligomers of a family of peptides derived from the β-amyloid peptide (Aβ). We had previously reported the X-ray crystallographic structures of the oligomers and oligomer assemblies formed in the solid state by a macrocyclic β-sheet peptide containing the Aβ_15–23 nonapeptide. In the current study, we set out to determine its assembly in aqueous solution. In the solid state, macrocyclic β-sheet peptide <b>1</b> assembles to form hydrogen-bonded dimers that further assemble in a sandwich-like fashion to form tetramers through hydrophobic interactions between the faces bearing V₁₈ and F₂₀. In aqueous solution, macrocyclic β-sheet peptide <b>1</b> and homologue <b>2a</b> form hydrogen-bonded dimers that assemble to form tetramers through hydrophobic interactions between the faces bearing L₁₇, F₁₉, and A₂₁. In the solid state, the hydrogen-bonded dimers are antiparallel, and the β-strands are fully aligned, with residues 17–23 of one of the macrocycles aligned with residues 23–17 of the other. In solution, residues 17–23 of the hydrogen-bonded dimers are shifted out of alignment by two residues toward the C-termini. The two hydrogen-bonded dimers are nearly orthogonal in the solid state, while in solution the dimers are only slightly rotated. The differing morphology of the solution-state and solid-state tetramers is significant, because it may provide a glimpse into some of the structural bases for polymorphism among Aβ oligomers in Alzheimer’s disease

A Hydrophobic Surface Is Essential To Inhibit the Aggregation of a Tau-Protein-Derived Hexapeptide

This paper seeks to understand how a macrocyclic β-sheet peptide inhibits the aggregation of the tau-protein-derived peptide Ac-VQIVYK-NH₂ (AcPHF6). Previous studies established that macrocyclic β-sheet peptide <b>1</b> inhibits AcPHF6 aggregation, while the sequence isomer in which the lysine and leucine residues at positions R₆ and R₇ are swapped has little effect on AcPHF6 aggregation. The current studies find that positions R₁, R₃, and R₇ are especially sensitive to mutations. Reducing hydrophobicity at these positions substantially diminishes inhibition. Although position R₅ is not sensitive to mutations that reduce hydrophobicity, it is sensitive to mutations that increase hydrophobicity. Enhanced hydrophobicity at this position substantially enhances inhibition. These studies establish that the hydrophobic surface comprising residues R₁, R₃, and R₇ is crucial to the inhibition process and that the residue R₅, which shares this surface, is also important. Collectively, these findings demonstrate that hydrophobic surfaces between β-sheet layers are important in inhibiting amyloid aggregation

X‑ray Crystallographic Structure of Oligomers Formed by a Toxic β‑Hairpin Derived from α‑Synuclein: Trimers and Higher-Order Oligomers

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 <b>1a</b> 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 <b>1a</b> interacts with anionic lipid bilayer membranes, like oligomers of full-length α-synuclein. LDH and MTT assays demonstrate that peptide <b>1a</b> is toxic toward SH-SY5Y cells. Comparison of peptide <b>1a</b> to homologues suggests that this toxicity results from nonspecific interactions with the cell membrane. The oligomers formed by peptide <b>1a</b> are fundamentally different than the proposed models of the fibrils formed by α-synuclein and suggest that α-Syn_36–55, rather than the NAC, may nucleate oligomer formation