Probing differences among Aß oligomers with two triangular trimers derived from Aß.

The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.

ß-Hairpin Alignment Alters Oligomer Formation in Aß-Derived Peptides.

Amyloid-ß (Aß) forms heterogeneous oligomers, which are implicated in the pathogenesis of Alzheimer's disease (AD). Many Aß oligomers consist of ß-hairpin building blocks─Aß peptides in ß-hairpin conformations. ß-Hairpins of Aß can adopt a variety of alignments, but the role that ß-hairpin alignment plays in the formation and heterogeneity of Aß oligomers is poorly understood. To explore the effect of ß-hairpin alignment on the oligomerization of Aß peptides, we designed and studied two model peptides with two different ß-hairpin alignments. Peptides Aßm17-36 and Aßm17-35 mimic two different ß-hairpins that Aß can form, the Aß17-36 and Aß17-35 ß-hairpins, respectively. These hairpins are similar in composition but differ in hairpin alignment, altering the facial arrangements of the side chains of the residues that they contain. X-ray crystallography and SDS-PAGE demonstrate that the difference in facial arrangement between these peptides leads to distinct oligomer formation. In the crystal state, Aßm17-36 forms triangular trimers that further assemble to form hexamers, while Aßm17-35 forms tetrameric ß-barrels. In SDS-PAGE, Aßm17-36 assembles to form a ladder of oligomers, while Aßm17-35 either assembles to form a dimer or does not assemble at all. The differences in the behavior of Aßm17-36 and Aßm17-35 suggest ß-hairpin alignment as a source of the observed heterogeneity of Aß oligomers.

Supramolecular Interactions of Teixobactin Analogues in the Crystal State.

This Note presents the X-ray crystallographic structure of the N-methylated teixobactin analogue N-Me-d-Gln4,Lys10-teixobactin (1). Eight peptide molecules comprise the asymmetric unit, with each peptide molecule binding a chloride anion through hydrogen bonding with the amide NH group of residues 7, 8, 10, and 11. The peptide molecules form hydrogen-bonded antiparallel ß-sheet dimers in the crystal lattice, with residues 1-3 comprising the dimerization interface. The dimers further assemble end-to-end in the crystal lattice.

Synthesis and Stereochemical Determination of the Peptide Antibiotic Novo29.

This paper describes the synthesis and stereochemical determination of Novo29 (clovibactin), a new peptide antibiotic that is related to teixobactin and is active against Gram-positive bacteria. Novo29 is an eight-residue depsipeptide that contains the noncanonical amino acid hydroxyasparagine of hitherto undetermined stereochemistry in a macrolactone ring. The amino acid building blocks Fmoc-(2R,3R)-hydroxyasparagine-OH and Fmoc-(2R,3S)-hydroxyasparagine-OH were synthesized from (R,R)- and (S,S)-diethyl tartrate. Novo29 and epi-Novo29 were then prepared by solid-phase peptide synthesis using these building blocks. Correlation with an authentic sample of Novo29 through 1H NMR spectroscopy, LC-MS, and in vitro antibiotic activity established that Novo29 contains (2R,3R)-hydroxyasparagine. X-ray crystallography reveals that epi-Novo29 adopts an amphiphilic conformation, with a hydrophobic surface and a hydrophilic surface. Four sets of epi-Novo29 molecules pack in the crystal lattice to form a hydrophobic core. The macrolactone ring adopts a conformation in which the main-chain amide NH groups converge to create a cavity, which binds ordered water and acetate anion. The amphiphilic conformation of epi-Novo29 is reminiscent of the amphiphilic conformation adopted by the related antibiotic teixobactin and its derivatives, which contains a hydrophobic surface that interacts with the lipids of the bacterial cell membrane and a hydrophilic surface that interacts with the aqueous environment. Molecular modeling suggests that Novo29 can adopt an amphiphilic conformation similar to teixobactin, suggesting that Novo29 may interact with bacteria in a similar fashion to teixobactin.

Effects of Familial Alzheimer's Disease Mutations on the Assembly of a ß-Hairpin Peptide Derived from Aß16-36.

Familial Alzheimer's disease (FAD) is associated with mutations in the ß-amyloid peptide (Aß) or the amyloid precursor protein (APP). FAD mutations of Aß were incorporated into a macrocyclic peptide that mimics a ß-hairpin to study FAD point mutations K16N, A21G, E22Δ, E22G, E22Q, E22K, and L34V and their effect on assembly, membrane destabilization, and cytotoxicity. The X-ray crystallographic structures of the four E22 mutant peptides reveal that the peptides assemble to form the same compact hexamer. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments reveal that the mutant FAD peptides assemble as trimers or hexamers, with peptides that have greater positive charge assembling as more stable hexamers. Mutations that increase the positive charge also increase the cytotoxicity of the peptides and their propensity to destabilize lipid membranes.

A Disulfide-Stabilized Aß that Forms Dimers but Does Not Form Fibrils.

Aß dimers are a basic building block of many larger Aß oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer's disease. Homogeneous Aß dimers are difficult to prepare, characterize, and study because Aß forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces AßC18C33 as a disulfide-stabilized analogue of Aß42 that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The AßC18C33 peptide is readily expressed in Escherichia coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that AßC18C33 forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric (MS) studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that AßC18C33 adopts a ß-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces ß-hairpin folding and dimer formation in lipid environments. Thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM) studies indicate that AßC18C33 does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aß42 tetramer (PDB: 6RHY) provides a working model for the AßC18C33 dimer, in which two ß-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel ß-sheet. It is anticipated that AßC18C33 will serve as a stable, nonfibrilizing, and noncovalent Aß dimer model for amyloid and Alzheimer's disease research.

Expression of N-Terminal Cysteine Aß42 and Conjugation to Generate Fluorescent and Biotinylated Aß42.

Fluorescent derivatives of the ß-amyloid peptides (Aß) are valuable tools for studying the interactions of Aß with cells. Facile access to labeled expressed Aß offers the promise of Aß with greater sequence and stereochemical integrity, without impurities from amino acid deletion and epimerization. Here, we report methods for the expression of Aß42 with an N-terminal cysteine residue, Aß(C1-42), and its conjugation to generate Aß42 bearing fluorophores or biotin. The methods rely on the hitherto unrecognized observation that expression of the Aß(MC1-42) gene yields the Aß(C1-42) peptide, because the N-terminal methionine is endogenously excised by Escherichia coli. Conjugation of Aß(C1-42) with maleimide-functionalized fluorophores or biotin affords the N-terminally labeled Aß42. The expression affords ∼14 mg of N-terminal cysteine Aß from 1 L of bacterial culture. Subsequent conjugation affords ∼3 mg of labeled Aß from 1 L of bacterial culture with minimal cost for labeling reagents. High-performance liquid chromatography analysis indicates the N-terminal cysteine Aß to be >97% pure and labeled Aß peptides to be 94-97% pure. Biophysical studies show that the labeled Aß peptides behave like unlabeled Aß and suggest that labeling of the N-terminus does not substantially alter the properties of the Aß. We further demonstrate applications of the fluorophore-labeled Aß peptides by using fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. We anticipate that these methods will provide researchers convenient access to useful N-terminally labeled Aß, as well as Aß with an N-terminal cysteine that enables further functionalization.

Phenylalanine Mutation to Cyclohexylalanine Facilitates Triangular Trimer Formation by ß-Hairpins Derived from Aß.

Oligomers of the ß-amyloid peptide, Aß, play a central role in the pathogenesis and progression of Alzheimer's disease. Trimers and higher-order oligomers composed of trimers are thought to be the most neurotoxic Aß oligomers. To gain insights into the structure and assembly of Aß oligomers, our laboratory has previously designed and synthesized macrocyclic peptides derived from Aß17-23 and Aß30-36 that fold to form ß-hairpins and assemble to form trimers. In this study, we found that mutating Phe20 to cyclohexylalanine (Cha) in macrocyclic Aß-derived peptides promotes crystallization of an Aß-derived peptide containing the Aß24-29 loop (peptide 3F20Cha) and permits elucidation of its structure and assembly by X-ray crystallography. X-ray crystallography shows that peptide 3F20Cha forms a hexamer. X-ray crystallography and SDS-PAGE further show that trimer 4F20Cha, a covalently stabilized trimer derived from peptide 3F20Cha, forms a dodecamer. Size exclusion chromatography shows that trimer 4F20Cha forms higher-order assemblies in solution. Trimer 4F20Cha exhibits cytotoxicity against the neuroblastoma cell line SH-SY5Y. These studies demonstrate the use of the F20Cha mutation to further stabilize oligomers of Aß-derived peptides that contain more of the native sequence and thus better mimic the oligomers formed by full-length Aß.

Effects of N-Terminal Residues on the Assembly of Constrained ß-Hairpin Peptides Derived from Aß.

This paper describes the synthesis, solution-phase biophysical studies, and X-ray crystallographic structures of hexamers formed by macrocyclic ß-hairpin peptides derived from the central and C-terminal regions of Aß, which bear "tails" derived from the N-terminus of Aß. Soluble oligomers of the ß-amyloid peptide, Aß, are thought to be the synaptotoxic species responsible for neurodegeneration in Alzheimer's disease. Over the last 20 years, evidence has accumulated that implicates the N-terminus of Aß as a region that may initiate the formation of damaging oligomeric species. We previously studied, in our laboratory, macrocyclic ß-hairpin peptides derived from Aß16-22 and Aß30-36, capable of forming hexamers that can be observed by X-ray crystallography and SDS-PAGE. To better mimic oligomers of full length Aß, we use an orthogonal protecting group strategy during the synthesis to append residues from Aß1-14 to the parent macrocyclic ß-hairpin peptide 1, which comprises Aß16-22 and Aß30-36. The N-terminally extended peptides N+1, N+2, N+4, N+6, N+8, N+10, N+12, and N+14 assemble to form dimers, trimers, and hexamers in solution-phase studies. X-ray crystallography reveals that peptide N+1 assembles to form a hexamer that is composed of dimers and trimers. These observations are consistent with a model in which the assembly of Aß oligomers is driven by hydrogen bonding and hydrophobic packing of the residues from the central and C-terminal regions, with the N-terminus of Aß accommodated by the oligomers as an unstructured tail.

Elucidating the Structures of Amyloid Oligomers with Macrocyclic ß-Hairpin Peptides: Insights into Alzheimer's Disease and Other Amyloid Diseases.

In 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-ß" structure in 1968. Further efforts led to the discovery of the ß-amyloid peptide, Aß, 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 Aß, 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. ß-Sheets are the building blocks of amyloid fibrils and oligomers. Amyloid fibrils generally consist of extended networks of parallel ß-sheets. Amyloid oligomers appear to be more compact enclosed structures, some of which are thought to be composed of antiparallel ß-sheets comprising ß-hairpins. ß-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 ß-sheets that are designed to mimic ß-hairpins formed by amyloidogenic peptides and proteins. The ß-hairpin mimics contain two ß-strand peptide fragments linked together at their N- and C-termini by two δ-linked ornithine turn mimics to create a macrocycle. An N-methyl group is installed on one of the ß-strands to prevent uncontrolled aggregation. These design features facilitate crystallization of the ß-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 ß-hairpin mimics derived from Aß, α-synuclein, and ß2-microglobulin. Out of these three amyloidogenic peptides and proteins, the Aß ß-hairpin mimics have provided the most insight into amyloid oligomers. Our studies have revealed a previously undiscovered mode of self-assembly, whereby three Aß ß-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 ß-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.

An Efficient Method for the Expression and Purification of Aß(M1-42).

Advances in amyloid research rely on improved access to the ß-amyloid peptide, Aß. N-Terminal methionine-extended Aß, Aß(M1-42), is a readily expressed and widely used form of Aß with properties comparable to those of the natural Aß(1-42) peptide. Expression of Aß(M1-42) is simple to execute and avoids an expensive and often difficult enzymatic cleavage step associated with expression and isolation of Aß(1-42). This paper reports an efficient method for the expression and purification of Aß(M1-42) and 15N-labeled Aß(M1-42). This method affords the pure peptide at ∼19 mg/L of bacterial culture through simple and inexpensive steps in 3 days. This paper also reports a simple method for the construction of recombinant plasmids and the expression and purification of Aß(M1-42) peptides containing familial mutations. We anticipate that these methods will enable experiments that would otherwise be hindered by insufficient access to Aß.

Controlling the Oligomerization State of Aß-Derived Peptides with Light.

A key challenge in studying the biological and biophysical properties of amyloid-forming peptides is that they assemble to form heterogeneous mixtures of soluble oligomers and insoluble fibrils. Photolabile protecting groups have emerged as tools to control the properties of biomolecules with light. Blocking intermolecular hydrogen bonds that stabilize amyloid oligomers provides a general strategy to control the biological and biophysical properties of amyloid-forming peptides. In this paper we describe the design, synthesis, and characterization of macrocyclic ß-hairpin peptides that are derived from amyloidogenic peptides and contain the N-2-nitrobenzyl photolabile protecting group. Each peptide contains two heptapeptide segments from Aß16-36 or Aß17-36 constrained into ß-hairpins. The N-2-nitrobenzyl group is appended to the amide backbone of Gly33 to disrupt the oligomerization of the peptides by disrupting intermolecular hydrogen bonds. X-ray crystallography reveals that N-2-nitrobenzyl groups can either block assembly into discrete oligomers or permit formation of trimers, hexamers, and dodecamers. Photolysis of the N-2-nitrobenzyl groups with long-wave UV light unmasks the amide backbone and alters the assembly and the biological properties of the macrocyclic ß-hairpin peptides. SDS-PAGE studies show that removing the N-2-nitrobenzyl groups alters the assembly of the peptides. MTT conversion and LDH release assays show that decaging the peptides induces cytotoxicity. Circular dichroism studies and dye leakage assays with liposomes reveal that decaging modulates interactions of the peptides with lipid bilayers. Collectively, these studies demonstrate that incorporating N-2-nitrobenzyl groups into macrocyclic ß-hairpin peptides provides a new strategy to probe the structures and the biological properties of amyloid oligomers.

Repurposing Triphenylmethane Dyes to Bind to Trimers Derived from Aß.

Soluble oligomers of the ß-amyloid peptide, Aß, 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 Aß oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aß17-36. 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 Phe19, Phe20, and Ile31 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 Phe20 side chains and is clasped by the Ile31 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 Aß can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aß.

A Hexamer of a Peptide Derived from Aß16-36.

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 ß-amyloid peptide Aß are especially important in the progression of Alzheimer's disease. In many Aß oligomers, the Aß monomer components are thought to adopt a ß-hairpin conformation. This paper describes the design and study of a macrocyclic ß-hairpin peptide derived from Aß16-36. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography studies show that the Aß16-36 ß-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 Aß16-36 ß-hairpin peptide are toxic toward neuronally derived SH-SY5Y cells. Replica-exchange molecular dynamics demonstrates that the hexamer can accommodate full-length Aß. These findings expand our understanding of the structure, solution-phase behavior, and biological activity of Aß oligomers and may offer insights into the molecular basis of Alzheimer's disease.

Stabilization, Assembly, and Toxicity of Trimers Derived from Aß.

Oligomers of the ß-amyloid peptide Aß have emerged as important contributors to neurodegeneration in Alzheimer's disease. Mounting evidence suggests that Aß 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 Aß. 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 Aß, 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 Aß ß-hairpins and may offer a deeper understanding of the molecular basis of Alzheimer's disease.

A Tetramer Derived from Islet Amyloid Polypeptide.

Aggregation of the islet amyloid polypeptide (IAPP) to form fibrils and oligomers is important in the progression of type 2 diabetes. This article describes X-ray crystallographic and solution-state NMR studies of peptides derived from residues 11-17 of IAPP that assemble to form tetramers. Incorporation of residues 11-17 of IAPP (RLANFLV) into a macrocyclic ß-sheet peptide results in a monomeric peptide that does not self-assemble to form oligomers. Mutation of Arg11 to the uncharged isostere citrulline gives peptide homologues that assemble to form tetramers in both the crystal state and in aqueous solution. The tetramers consist of hydrogen-bonded dimers that sandwich together through hydrophobic interactions. The tetramers share several features with structures reported for IAPP fibrils and demonstrate the importance of hydrogen bonding and hydrophobic interactions in the oligomerization of IAPP-derived peptides.

X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aß17-36 ß-Hairpin.

High-resolution structures of oligomers formed by the ß-amyloid peptide Aß 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 Aß. The development of a peptide in which Aß17-36 is stabilized as a ß-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. Two covalent constraints act in tandem to stabilize the Aß17-36 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 ß-hairpin, which assembles hierarchically in the crystal lattice. Three ß-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 Aß transitions from an unfolded monomer to a folded ß-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 Structures of Oligomers of Peptides Derived from ß2-Microglobulin.

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 ß2-microglobulin (ß2m). Each macrocyclic peptide contains the heptapeptide sequence ß2m63-69 and a second heptapeptide sequence containing an N-methyl amino acid. These peptides form ß-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 ß-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.