Switching in amyloid structure within individual fibrils: Implication for strain adaptation, species barrier and strain classification

Amyloid fibrils are highly ordered crystal-like structures. It is generally assumed that individual amyloid fibrils consist of conformationally uniform cross-β-sheet structures that enable the amyloids to replicate their individual conformations via a template-dependent mechanism. Recent studies revealed that amyloids are capable of accommodating a global conformational switch from one amyloid strain to another within individual fibrils. The current review highlights the high adaptation potential of amyloid structures and discusses the implication of these findings for several emerging issues including prion strain adaptation (i.e. gradual change in strain structure). It also proposes that the catalytic activity of an amyloid structure should be separated from its templating effect, and raises the question of strain classification according to their promiscuous or species-specific nature.     

Globular Tetramers of β2-Microglobulin Assemble into Elaborate Amyloid Fibrils

Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-β-strands and have revealed some details of local β-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of three-dimensional structures of amyloid fibrils formed from full-length β₂-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold.     

Heparin binds 8 kDa gelsolin cross-β-sheet oligomers and accelerates amyloidogenesis by hastening fibril extension

Glycosaminoglycans (GAGs) are highly sulfated linear polysaccharides prevalent in the extracellular matrix, and they associate with virtually all amyloid deposits in vivo. GAGs accelerate the aggregation of many amyloidogenic peptides in vitro, but little mechanistic evidence is available to explain why. Herein, spectroscopic methods demonstrate that GAGs do not affect the secondary structure of the monomeric 8 kDa amyloidogenic fragment of human plasma gelsolin. Moreover, monomerized 8 kDa gelsolin does not bind to heparin under physiological conditions. In contrast, 8 kDa gelsolin cross-β-sheet oligomers and amyloid fibrils bind strongly to heparin, apparently because of electrostatic interactions between the negatively charged polysaccharide and a positively charged region of the 8 kDa gelsolin assemblies. Our observations are consistent with a scaffolding mechanism whereby cross-β-sheet oligomers, upon formation, bind to GAGs, accelerating the fibril extension phase of amyloidogenesis, possibly by concentrating and orienting the oligomers to more efficiently form amyloid fibrils. Notably, heparin decreases the 8 kDa gelsolin concentration necessary for amyloid fibril formation, likely a consequence of fibril stabilization through heparin binding. Because GAG overexpression, which is common in amyloidosis, may represent a strategy for minimizing cross-β-sheet oligomer toxicity by transforming them into amyloid fibrils, the mechanism described herein for GAG-mediated acceleration of 8 kDa gelsolin amyloidogenesis provides a starting point for therapeutic strategy development. The addition of GAG mimetics, small molecule sulfonates shown to reduce the amyloid load in animal models of amyloidosis, to a heparin-accelerated 8 kDa gelsolin aggregation reaction mixture neither significantly alters the rate of amyloidogenesis nor prevents oligomers from binding to GAGs, calling into question their commonly accepted mechanism.     

Aβ(1-40) Forms Five Distinct Amyloid Structures whose β-Sheet Contents and Fibril Stabilities Are Correlated

The ability of a single polypeptide sequence to grow into multiple stable amyloid fibrils sets these aggregates apart from most native globular proteins. The existence of multiple amyloid forms is the basis for strain effects in yeast prion biology, and might contribute to variations in Alzheimer's disease pathology. However, the structural basis for amyloid polymorphism is poorly understood. We report here five structurally distinct fibrillar aggregates of the Alzheimer's plaque peptide Aβ(1-40), as well as a non-fibrillar aggregate induced by Zn²⁺. Each of these conformational forms exhibits a unique profile of physical properties, and all the fibrillar forms breed true in elongation reactions under a common set of growth conditions. Consistent with their defining cross-β structure, we find that in this series the amyloid fibrils containing more extensive β-sheet exhibit greater stability. At the same time, side chain packing outside of the β-sheet regions contributes to stability, and to differences of stability between polymorphic forms. Stability comparison is facilitated by the unique feature that the free energy of the monomer (equivalent to the unfolded state in a protein folding reaction) does not vary, and hence can be ignored, in the comparison of ΔG° of elongation values for each polymorphic fibril obtained under a single set of conditions.     

A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple ‘letter-G-like’ β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops.     

Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.     

The Molecular Organization of the Fungal Prion HET-s in Its Amyloid Form

The prion hypothesis states that it is solely the three-dimensional structure of the polypeptide chain that distinguishes the prion and nonprion forms of the protein. For HET-s, the atomic-resolution structure of the isolated prion domain HET-s(218-289), consisting of a highly ordered triangular cross-β arrangement, is known. Here we present a solid-state NMR study of fibrils of the full-length HET-s prion in which we compare their spectra with spectra from isolated C-terminal prion domain fibrils and the crystalline N-terminal globular domain HET-s(1-227). The spectra reveal unequivocally that the highly ordered structure of the isolated prion domain HET-s(218-289) is conserved in the context of the full-length fibrils investigated here. However, the globular domain loses much of its tertiary structure while partly retaining its secondary structure, thus exhibiting behavior reminiscent of a molten globule. Flexible residues that may constitute the linker connecting the two domains are detected using INEPT (insensitive nuclei enhanced by polarization transfer) spectroscopy. Based on our data, we propose a structural model that is in line with a general model developed for amyloid fibrils built from a cross-β core decorated with globular domains. The loss of structure in the HET-s globular domain sharply contrasts with the behavior observed for fibrils of Ure2p and suggests that there is considerable structural diversity in the fibrils of globular-domain-containing prions despite their similar appearances at the microscopic level.     

The Common Architecture of Cross-β Amyloid

Amyloid fibril deposition is central to the pathology of more than 30 unrelated diseases including Alzheimer's disease and Type 2 diabetes. It is generally accepted that amyloid fibrils share common structural features despite each disease being characterised by the deposition of an unrelated protein or peptide. The structure of amyloid fibrils has been studied using X-ray fibre diffraction and crystallography, solid-state NMR and electron paramagnetic resonance, and many different, sometimes opposing, models have been suggested. Many of these models are based on the original interpretation of the cross-β diffraction pattern for cross-β silk in which β-strands run perpendicular to the fibre axis, although alternative models include β-helices and natively structured proteins. Here, we have analysed opposing model structures and examined the necessary structural elements within the amyloid core structure, as well as producing idealised models to test the limits of the core conformation. Our work supports the view that amyloid fibrils share a number of common structural features, resulting in characteristic diffraction patterns. This pattern may be satisfied by structures in which the strands align close to perpendicular to the fibre axis and are regularly arranged to form β-sheet ribbons. Furthermore, the fibril structure contains several β-sheets that associate via side-chain packing to form the final protofilament structure.     

Comparative analysis of 13C chemical shifts of β-sheet amyloid proteins and outer membrane proteins

Cross-β amyloid fibrils and membrane-bound β-barrels are two important classes of β-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the ¹³C chemical shifts of 17 amyloid proteins and 7 β-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 β-sheet residues in amyloid fibrils and 521 β-sheet residues in β-barrel membrane proteins. The ¹³C chemical shifts are shown in 2D ¹³C–¹³C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in β-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and β-barrels and compare the sidechain rotameric populations. The ¹³C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in β-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in β-barrels. These trends can be explained by steric zipper interactions between β-sheet planes in cross-β fibrils, and by the interactions of β-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and β-barrels based principally on NMR chemical shifts.

     

The effect of lysozyme amyloid fibrils on cytochrome c–lipid interactions

Protein polymerization into ordered fibrillar structures (amyloid fibrils) is currently associated with a range of pathological conditions. Recent studies clearly indicate that amyloid cytotoxicity is provoked by a continuum of cross-β-sheet aggregates including mature fibrils. In view of the possible diversity of cytotoxicity mechanisms, the present study addressed the question of whether protein conversion into amyloid fibrils can modify its competitive membrane adsorption behavior. Using a combination of resonance energy transfer, dynamic light scattering and fluorescence quenching techniques, the competitive binding of either monomeric or polymerized lysozyme, and cytochrome c to the model lipid membranes composed of phosphatidylcholine mixtures with varying proportions of phosphatidylglycerol, phosphatidylserine or cardiolipin has been studied. The ability of fibrillar lysozyme to induce dissociation of cytochrome c from the membrane binding sites proved to be markedly stronger than that of its monomeric counterpart, with desorption process displaying cooperativity features upon increasing the charge of lipid bilayer. The decreased efficiency of tryptophan fluorescence quenching by acrylamide and short-wavelength shift of emission maximum observed upon membrane binding of lysozyme fibrils were rationalized in terms of fluorophore transfer into interfacial bilayer region. It is hypothesized that electrostatic interactions play predominant role in determining the lipid-associating and competitive abilities of fibrillar lysozyme.     

Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size

Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-β-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions—perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including αB-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.     

Bovine Insulin Filaments Induced by Reducing Disulfide Bonds Show a Different Morphology, Secondary Structure, and Cell Toxicity from Intact Insulin Amyloid Fibrils

Amyloid fibrils are associated with more than 20 diseases, including Alzheimer's disease and type II diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed due to partial reduction of S-S bonds in insulin molecules, differ from intact insulin fibrils in terms of their secondary structure. The insulin filaments were determined to have an antiparallel β-sheet structure, whereas the insulin fibrils have a parallel β-sheet structure. Of importance, the cell toxicity of the insulin filaments was remarkably lower than that of the insulin fibrils. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The remarkably low toxicity of the filamentous structure should shed new light on possible pharmacological approaches to the various diseases caused by amyloid fibrils.     

Amino acid sequence determinants and molecular chaperones in amyloid fibril formation

Amyloid consists of cross-β-sheet fibrils and is associated with about 25 human diseases, including several neurodegenerative diseases, systemic and localized amyloidoses and type II diabetes mellitus. Amyloid-forming proteins differ in structures and sequences, and it is to a large extent unknown what makes them convert from their native conformations into amyloid. In this review, current understanding of amino acid sequence determinants and the effects of molecular chaperones on amyloid formation are discussed. Studies of the nonpolar, transmembrane surfactant protein C (SP-C) have revealed amino acid sequence features that determine its amyloid fibril formation, features that are also found in the amyloid β-peptide in Alzheimer’s disease and the prion protein. Moreover, a proprotein chaperone domain (CTC_Brichos) that prevents amyloid-like aggregation during proSP-C biosynthesis can prevent fibril formation also of other amyloidogenic proteins.     

Pattern recognition with a fibril-specific antibody fragment reveals the surface variability of natural amyloid fibrils

Amyloid immunotherapy has led to the rise of antibodies, which target amyloid fibrils or structural precursors of fibrils, based on their specific conformational properties. Recently, we reported the biotechnological generation of the B10 antibody fragment, which provides conformation-specific binding to amyloid fibrils. B10 strongly interacts with fibrils from Alzheimer's β amyloid (Aβ) peptide, while disaggregated Aβ peptide or Aβ oligomers are not explicitly recognized. B10 also enables poly-amyloid-specific binding and recognizes amyloid fibrils derived from different types of amyloidosis or different polypeptide chains. Based on our current data, however, we find that B10 does not recognize all tested amyloid fibrils and amyloid tissue deposits. It also does not specifically interact with intrinsically unfolded polypeptide chains or globular proteins even if the latter encompass high β-sheet content or β-solenoid domains. By contrast, B10 binds amyloid fibrils from d-amino acid or l-amino acid peptides and non-proteinaceous biopolymers with highly regular and anionic surface properties, such as heparin and DNA. These data establish that B10 binding does not depend on an amyloid-specific or protein-specific backbone structure. Instead, it involves the recognition of a highly regular and anionic surface pattern. This specificity mechanism is conserved in nature and occurs also within a group of natural amyloid receptors from the innate immune system, the pattern recognition receptors. Our data illuminate the structural diversity of naturally occurring amyloid scaffolds and enable the discrimination of distinct fibril populations in vitro and within diseased tissues.     

Amyloid fibril recognition with the conformational B10 antibody fragment depends on electrostatic interactions

Amyloid fibrils are naturally occurring polypeptide scaffolds with considerable importance for human health and disease. These supermolecular assemblies are β-sheet rich and characterized by a high structural order. Clinical diagnosis and emerging therapeutic strategies of amyloid-dependent diseases, such as Alzheimer's, rely on the specific recognition of amyloid structures by other molecules. Recently, we generated the B10 antibody fragment, which selectively binds to Alzheimer's Aβ(1-40) amyloid fibrils but does not explicitly recognize other protein conformers, such as oligomers and disaggregated Aβ peptide. B10 presents poly-amyloid specific binding and interacts with fibrillar structures consisting of different polypeptide chains. To determine the molecular basis behind its specificity, we have analyzed the molecular properties of B10 with a battery of biochemical and biophysical techniques, ranging from X-ray crystallography to chemical modification studies. We find that fibril recognition depends on positively charged residues within the B10 antigen binding site. Mutation of these basic residues into alanine potently impairs fibril binding, and reduced B10-fibril interactions are also observed when the fibril carboxyl groups are covalently masked by a chemical modification approach. These data imply that the B10 conformational specificity for amyloid fibrils depends upon specific electrostatic interactions with an acidic moiety, which is common to different amyloid fibrils.     

Intrinsic determinants of Aβ(12-24) pH-dependent self-assembly revealed by combined computational and experimental studies

The propensity of amyloid-β (Aβ) peptide to self-assemble into highly ordered amyloid structures lies at the core of their accumulation in the brain during Alzheimer's disease. By using all-atom explicit solvent replica exchange molecular dynamics simulations, we elucidated at the atomic level the intrinsic determinants of the pH-dependent dimerization of the central hydrophobic segment Aβ(12-24) and related these with the propensity to form amyloid fibrils measured by experimental tools such as atomic force microscopy and fluorescence. The process of Aβ(12-24) dimerization was evaluated in terms of free energy landscape, side-chain two-dimensional contact probability maps, β-sheet registries, potential mean force as a function of inter-chain distances, secondary structure development and radial solvation distributions. We showed that dimerization is a key event in Aβ(12-24) amyloid formation; it is highly prompted in the order of pH 5.0>2.9>8.4 and determines further amyloid growth. The dimerization is governed by a dynamic interplay of hydrophobic, electrostatic and solvation interactions permitting some variability of β-sheets at each pH. These results provide atomistic insight into the complex process of molecular recognition detrimental for amyloid growth and pave the way for better understanding of the molecular basis of amyloid diseases.     

Amyloid Fibril-Like Structure Underlies the Aggregate Structure across the pH Range for β-Lactoglobulin

The protein β-lactoglobulin aggregates into two apparently distinct forms under different conditions: amyloid fibrils at pH values away from the isoelectric point, and spherical aggregates near it. To understand this apparent dichotomy in behavior, we studied the internal structure of the spherical aggregates by employing a range of biophysical approaches. Fourier transform infrared studies show the aggregates have a high β-sheet content that is distinct from the native β-lactoglobulin structure. The structures also bind the amyloidophilic dye thioflavin-T, and wide-angle x-ray diffraction showed reflections corresponding to spacings typically observed for amyloid fibrils composed of β-lactoglobulin. Combined with small-angle x-ray scattering data indicating the presence of one-dimensional linear aggregates at the molecular level, these findings indicate strongly that the aggregates contain amyloid-like substructure. Incubation of β-lactoglobulin at pH values increasingly removed from the isoelectric point resulted in the increasing appearance of fibrillar species, rather than spherical species shown by electron microscopy. Taken together, these results suggest that amyloid-like β-sheet structures underlie protein aggregation over a much broader range of conditions than previously believed. Furthermore, the results suggest that there is a continuum of β-sheet structure of varying regularity underlying the aggregate morphology, from very regular amyloid fibrils at high charge to short stretches of amyloid-like fibrils that associate together randomly to form spherical particles at low net charge.     

Chiral bifurcation in aggregating insulin: an induced circular dichroism study

The structural unambiguity of folding is lost when disordered protein molecules convert into β-sheet-rich fibrils. The resulting polymorphism of protein aggregates has been studied in the context of its biomedical consequences. Events underlying the conformational variance of amyloid fibrils, as well as physicochemical boundaries between folding and misfolding pathways, remain obscure. Bifurcation and chiral mesoscopic-scale organization of amyloid fibrils are new aspects of protein misfolding. Here we characterize bifurcation events accompanying insulin fibrillation upon intensive vortexing. Upon agitation, two types of insulin fibrils with opposite chiral senses are formed; however, predominance of either species is only stochastically determined. The uncertainty of fibrils’ chiral sense holds only for fibrils grown within the physiological temperature range, while above 50 °C, the bifurcation is no longer observed—fibrils’ chiral moieties become uniformly biased towards ligand probes, as revealed by the extrinsic Cotton effect of thioflavin T, Congo red, and molecular iodine. According to transmission electron microscopy and scanning electron microscopy data, chiral variants of insulin fibrils consist of fibrous superstructures, distinct from spherulites, formed by the protein in nonagitated solutions. Gradual dissociation of the fibrils in the presence of dimethyl sulfoxide is noncooperative and can be resolved into three distinct phases: decay of the higher-order chiral structures, breakdown of fibrils, and unfolding of intermolecular β-sheet. The chiral aggregates are also destabilized by elution of NaCl implying that Debye screening of charged β-sheets provided by chloride counterions is needed for sustaining their kinetic stability. At elevated temperatures, cross-seeding of agitated insulin samples with preformed fibrils revealed a chiral conflict that prevented the passing of structural features of mother seeds to daughter fibrils in a manner typical of amyloid “strains.”     

Aβ(1-40) Fibril Polymorphism Implies Diverse Interaction Patterns in Amyloid Fibrils

Amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer's disease, Creutzfeldt-Jakob and type II diabetes. Alzheimer's amyloid fibrils consist of amyloid-β (Aβ) peptide and occur in a range of structurally different fibril morphologies. The structural characteristics of 12 single Aβ(1-40) amyloid fibrils, all formed under the same solution conditions, were determined by electron cryo-microscopy and three-dimensional reconstruction. The majority of analyzed fibrils form a range of morphologies that show almost continuously altering structural properties. The observed fibril polymorphism implies that amyloid formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.