Ryan K. Spencer

X-ray Crystallographic Structures of Trimers and Higher-Order Oligomeric Assemblies of a Peptide Derived from Aβ17–36

A peptide derived from Aβ17–36 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.

A Fibril-Like Assembly of Oligomers of a Peptide Derived from β-Amyloid

A macrocyclic β-sheet peptide containing two nonapeptide segments based on Aβ15–23 (QKLVFFAED) forms fibril-like assemblies of oligomers in the solid state. The X-ray crystallographic structure of macrocyclic β-sheet peptide 3 was determined at 1.75 Å resolution. The macrocycle forms hydrogen-bonded dimers, which further assemble along the fibril axis in a fashion resembling a herringbone pattern. The extended β-sheet comprising the dimers is laminated against a second layer of dimers through hydrophobic interactions to form a fibril-like assembly that runs the length of the crystal lattice. The second layer is offset by one monomer subunit, so that the fibril-like assembly is composed of partially overlapping dimers, rather than discrete tetramers. In aqueous solution, macrocyclic β-sheet 3 and homologues 4 and 5 form discrete tetramers, rather than extended fibril-like assemblies. The fibril-like assemblies of oligomers formed in the solid state by macrocyclic β-sheet 3 represent a new mode of supramolecular assembly not previously observed for the amyloidogenic central region of Aβ. The structures observed at atomic resolution for this peptide model system may offer insights into the structures of oligomers and oligomer assemblies formed by full-length Aβ and may provide a window into the propagation and replication of amyloid oligomers.

X-ray Crystallographic Structures of Oligomers of Peptides Derived from β2-Microglobulin.

Amyloid diseases such as Alzheimers disease, Parkinsons 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 β2-microglobulin (β2m). Each macrocyclic peptide contains the heptapeptide sequence β2m63-69 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 dodecamer-forming 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.

X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin

High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are needed to understand the molecular basis of Alzheimer’s disease and develop therapies. This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from Aβ. The development of a peptide in which Aβ17–36 is stabilized as a β-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. An N-methyl group at position 33 blocks uncontrolled aggregation. The peptide readily crystallizes as a folded β-hairpin, which assembles hierarchically in the crystal lattice. Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. This model may provide a better understanding of the molecular basis of Alzheimer’s disease at atomic resolution.

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 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.

Heterodivalent Linked Macrocyclic β-Sheets with Enhanced Activity against Aβ Aggregation: Two Sites Are Better Than One

This paper reports a series of heterodivalent linked macrocyclic β-sheets 6 that are not only far more active against amyloid-β (Aβ) aggregation than their monovalent components 1a and 1b but also are dramatically more active than their homodivalent counterparts 4 and 5. The macrocyclic β-sheet components 1a and 1b comprise pentapeptides derived from the N- and C-terminal regions of Aβ and molecular template and turn units that enforce a β-sheet structure and block aggregation. Thioflavin T fluorescence assays show that heterodivalent linked macrocyclic β-sheets 6 delay Aβ(1-40) aggregation 6-8-fold at equimolar concentrations and substantially delay aggregation at substoichiometric concentrations, while homodivalent linked macrocyclic β-sheets 4 and 5 and monovalent macrocyclic β-sheets 1a and 1b only exhibit more modest effects at equimolar or greater concentrations. A model to explain these observations is proposed, in which the inhibitors bind to and stabilize the early β-structured Aβ oligomers and thus delay aggregation. In this model, heterodivalent linked macrocyclic β-sheets 6 bind to the β-structured oligomers more strongly, because N-terminal-derived component 1a can bind to the N-terminal-based core of the β-structured oligomers, while the C-terminal-derived component 1b can achieve additional interactions with the C-terminal region of Aβ. The enhanced activity of the heterodivalent compounds suggests that polyvalent inhibitors that can target multiple regions of amyloidogenic peptides and proteins are better than those that only target a single region.

Stabilization, Assembly, and Toxicity of Trimers Derived from Aβ

Oligomers of the β-amyloid peptide Aβ have emerged as important contributors to neurodegeneration in Alzheimer’s disease. Mounting evidence suggests that Aβ trimers and higher-order oligomers derived from trimers have special significance in the early stages of Alzheimer’s disease. Elucidating the structures of these trimers and higher-order oligomers is paramount for understanding their role in neurodegeneration. This paper describes the design, synthesis, X-ray crystallographic structures, and biophysical and biological properties of two stabilized trimers derived from the central and C-terminal regions of Aβ. These triangular trimers are stabilized through three disulfide cross-links between the monomer subunits. The X-ray crystallographic structures reveal that the stabilized trimers assemble hierarchically to form hexamers, dodecamers, and annular porelike structures. Solution-phase biophysical studies reveal that the stabilized trimers assemble in solution to form oligomers that recapitulate some of the higher-order assemblies observed crystallographically. The stabilized trimers share many of the biological characteristics of oligomers of full-length Aβ, including toxicity toward a neuronally derived human cell line, activation of caspase-3 mediated apoptosis, and reactivity with the oligomer-specific antibody A11. These studies support the biological significance of the triangular trimer assembly of Aβ β-hairpins and may offer a deeper understanding of the molecular basis of Alzheimer’s disease.

Sequence-Dependent Self-Assembly and Structural Diversity of Islet Amyloid Polypeptide-Derived β-Sheet Fibrils

Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. We anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.

X-ray Crystallographic Structure of a Compact Dodecamer from a Peptide Derived from Aβ16–36

The assembly of the β-amyloid peptide, Aβ, into soluble oligomers is associated with neurodegeneration in Alzheimers 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.

Oxygen K Edge Scattering from Bulk Comb Diblock Copolymer Reveals Extended, Ordered Backbones above Lamellar Order–Disorder Transition

The evolution of molecular morphology in bulk samples of comb diblock copolymer pNdc12-b-pNte21 across the lamellar order-disorder transition (ODT) is studied using resonant X-ray scattering at the oxygen K edge with the goal of determining whether the molecules remain extended or collapse above the ODT. The distinct spectral resonances of carbonyl oxygen on the backbone and ether oxygen in the pNte side chains combine with their different site symmetry within the molecule to yield strong differences in bulk structural sensitivity at all temperatures. Comparison with simple models for the disordered phase clearly reveals that disordering at the ODT corresponds to loss of positional order of molecules with extended backbones that retain orientational order, rather than backbone collapse into a locally isotropic disordered phase. This conclusion is facilitated directly by the distinct structural sensitivity at the two resonances. The roles of depolarized scattering in enhancing this sensitivity, and background fluorescence in limiting dynamic range in oxygen resonant scattering are discussed.