Teixobactin kills bacteria by a two-pronged attack on the cell envelope.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

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.

Amyloid-α Peptide Formed through Alternative Processing of the Amyloid Precursor Protein Attenuates Alzheimer's Amyloid-ß Toxicity via Cross-Chaperoning.

Amyloid aggregation is a key feature of Alzheimer's disease (AD) and a primary target for past and present therapeutic efforts. Recent research is making it increasingly clear that the heterogeneity of amyloid deposits, extending past the commonly targeted amyloid-ß (Aß), must be considered for successful therapy. We recently demonstrated that amyloid-α (Aα or p3), a C-terminal peptidic fragment of Aß, aggregates rapidly to form amyloids and can expedite the aggregation of Aß through seeding. Here, we advance the understanding of Aα biophysics and biology in several important ways. We report the first cryogenic electron microscopy (cryo-EM) structure of an Aα amyloid fibril, proving unambiguously that the peptide is fibrillogenic. We demonstrate that Aα induces Aß to form amyloid aggregates that are less toxic than pure Aß aggregates and use nuclear magnetic resonance spectroscopy (NMR) to provide insights into specific interactions between Aα and Aß in solution. This is the first evidence that Aα can coassemble with Aß and alter its biological effects at relatively low concentrations. Based on the above, we urge researchers in the field to re-examine the significance of Aα in AD.

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.

A turn for the worse: Aß ß-hairpins in Alzheimer's disease.

Amyloid-ß (Aß) oligomers are a cause of neurodegeneration in Alzheimer's disease (AD). These soluble aggregates of the Aß peptide have proven difficult to study due to their inherent metastability and heterogeneity. Strategies to isolate and stabilize homogenous Aß oligomer populations have emerged such as mutations, covalent cross-linking, and protein fusions. These strategies along with molecular dynamics simulations have provided a variety of proposed structures of Aß oligomers, many of which consist of molecules of Aß in ß-hairpin conformations. ß-Hairpins are intramolecular antiparallel ß-sheets composed of two ß-strands connected by a loop or turn. Three decades of research suggests that Aß peptides form several different ß-hairpin conformations, some of which are building blocks of toxic Aß oligomers. The insights from these studies are currently being used to design anti-Aß antibodies and vaccines to treat AD. Research suggests that antibody therapies designed to target oligomeric Aß may be more successful at treating AD than antibodies designed to target linear epitopes of Aß or fibrillar Aß. Aß ß-hairpins are good epitopes to use in antibody development to selectively target oligomeric Aß. This review summarizes the research on ß-hairpins in Aß peptides and discusses the relevance of this conformation in AD pathogenesis and drug development.

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.

An Improved Turn Structure for Inducing ß-Hairpin Formation in Peptides.

Although ß-hairpins are widespread in proteins, there is no tool to coax any small peptide to adopt a ß-hairpin conformation, regardless of sequence. Here, we report that δ-linked γ(R)-methyl-ornithine (δ MeOrn) provides an improved ß-turn template for inducing a ß-hairpin conformation in peptides. We developed a synthesis of protected δ MeOrn as a building block suitable for use in Fmoc-based solid-phase peptide synthesis. The synthesis begins with l-leucine and affords gram quantities of the Nα -Boc-Nδ -Fmoc-γ(R)-methyl-ornithine building block. X-ray crystallography confirms that the δ MeOrn turn unit adopts a folded structure in a macrocyclic ß-hairpin peptide. CD and NMR spectroscopy allow comparison of the δ MeOrn turn template to the δ-linked ornithine (δ Orn) turn template that we previously introduced and to the popular d-Pro-Gly turn template. These studies show that the folding of the δ MeOrn turn template is substantially better than that of δ Orn and is comparable to d-Pro-Gly.

Interpenetrating Cubes in the X-ray Crystallographic Structure of a Peptide Derived from Medin19-36.

Amyloidogenic peptides and proteins are rich sources of supramolecular assemblies. Sequences derived from well-known amyloids, including Aß, human islet amyloid polypeptide, and tau have been found to assemble as fibrils, nanosheets, ribbons, and nanotubes. The supramolecular assembly of medin, a 50-amino acid peptide that forms fibrillary deposits in aging human vasculature, has not been heavily investigated. In this work, we present an X-ray crystallographic structure of a cyclic ß-sheet peptide derived from the 19-36 region of medin that assembles to form interpenetrating cubes. The edge of each cube is composed of a single peptide, and each vertex is occupied by a divalent metal ion. This structure may be considered a metal-organic framework (MOF) containing a large peptide ligand. This work demonstrates that peptides containing Glu or Asp that are preorganized to adopt ß-hairpin structures can serve as ligands and assemble with metal ions to form MOFs.

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.

Anaphylaxis Induced by Peptide Coupling Agents: Lessons Learned from Repeated Exposure to HATU, HBTU, and HCTU.

Peptide coupling agents pose a special hazard because they are immune sensitizers. Here, we present a case study of anaphylaxis induced by three uronium coupling agents, HATU, HBTU, and HCTU, as a cautionary note for researchers who handle peptide coupling agents frequently. We also include recommendations for handling coupling agents more safely in the research laboratory.

Design, Synthesis, and Study of Lactam and Ring-Expanded Analogues of Teixobactin.

This paper describes the chemical synthesis, X-ray crystallographic structure, and antibiotic activity assay of lactam analogues of teixobactin and explores ring-expanded analogues of teixobactin with ß3-homo amino acids. Lactam analogues of teixobactin containing all four stereoisomers of aza-threonine at position 8 were synthesized on a solid support from commercially available stereoisomeric threonine derivatives. The threonine stereoisomers are converted to the diastereomeric aza-threonines by mesylation, azide displacement, and reduction during the synthesis. d-Aza-Thr8,Arg10-teixobactin exhibits 2-8-fold greater antibiotic activity than the corresponding macrolactone Arg10-teixobactin. Azateixobactin analogues containing other stereoisomers of aza-threonine are inactive. A dramatic 16-128-fold increase in the activity of teixobactin and teixobactin analogues is observed with the inclusion of 0.002% of the mild detergent polysorbate 80 in the MIC assay. The X-ray crystallographic structure of N-Me-d-Gln4,d-aza-Thr8,Arg10-teixobactin reveals an amphipathic hydrogen-bonded antiparallel ß-sheet dimer that binds chloride anions. In the binding site, the macrolactam amide NH groups of residues 8, 10, and 11, as well as the extra amide NH group of the lactam ring, hydrogen bond to the chloride anion. The teixobactin pharmacophore tolerates ring expansion of the 13-membered ring to 14-,15-, and 16-membered rings containing ß3-homo amino acids with retention of partial or full antibiotic activity.

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.

Structural Polymorphs Suggest Competing Pathways for the Formation of Amyloid Fibrils That Diverge from a Common Intermediate Species.

It is now recognized that many amyloid-forming proteins can associate into multiple fibril structures. Here, we use two-dimensional infrared spectroscopy to study two fibril polymorphs formed by human islet amyloid polypeptide (hIAPP or amylin), which is associated with type 2 diabetes. The polymorphs exhibit different degrees of structural organization near the loop region of hIAPP fibrils. The relative populations of these polymorphs are systematically altered by the presence of macrocyclic peptides which template ß-sheet formation at specific sections of the hIAPP sequence. These experiments are consistent with polymorphs that result from competing pathways for fibril formation and that the macrocycles bias hIAPP aggregation toward one pathway or the other. Another macrocyclic peptide that matches the loop region but extends the lag time leaves the relative populations of the polymorphs unaltered, suggesting that the branching point for structural divergence occurs after the lag phase, when the oligomers convert into seeds that template fibril formation. Thus, we conclude that the structures of the polymorphs stem from restricting oligomers along diverging folding pathways, which has implications for drug inhibition, cytotoxicity, and the free energy landscape of hIAPP aggregation.