Molecular structure of amyloid fibrils: insights from solid-state NMR

Solid-state nuclear magnetic resonance (NMR) measurements have made major contributions to our understanding of the molecular structures of amyloid fibrils, including fibrils formed by the beta-amyloid peptide associated with Alzheimer's disease, by proteins associated with fungal prions, and by a variety of other polypeptides. Because solid-state NMR techniques can be used to determine interatomic distances (both intramolecular and intermolecular), place constraints on backbone and side-chain torsion angles, and identify tertiary and quaternary contacts, full molecular models for amyloid fibrils can be developed from solid-state NMR data, especially when supplemented by lower-resolution structural constraints from electron microscopy and other sources. In addition, solid-state NMR data can be used as experimental tests of various proposals and hypotheses regarding the mechanisms of amyloid formation, the nature of intermediate structures, and the common structural features within amyloid fibrils. This review introduces the basic experimental and conceptual principles behind solid-state NMR methods that are applicable to amyloid fibrils, reviews the information about amyloid structures that has been obtained to date with these methods, and discusses how solid-state NMR data provide insights into the molecular interactions that stabilize amyloid structures, the generic propensity of polypeptide chains to form amyloid fibrils, and a number of related issues that are of current interest in the amyloid field.     

Physical and structural basis for polymorphism in amyloid fibrils

As our understanding of the molecular structures of amyloid fibrils has matured over the past 15 years, it has become clear that, while amyloid fibrils do have well‐defined molecular structures, their molecular structures are not uniquely determined by the amino acid sequences of their constituent peptides and proteins. Self‐propagating molecular‐level polymorphism is a common phenomenon. This article reviews current information about amyloid fibril structures, variations in molecular structures that underlie amyloid polymorphism, and physical considerations that explain the development and persistence of amyloid polymorphism. Much of this information has been obtained through solid state nuclear magnetic resonance measurements. The biological significance of amyloid polymorphism is also discussed briefly. Although this article focuses primarily on studies of fibrils formed by amyloid‐β peptides, the same principles apply to many amyloid‐forming peptides and proteins.     

Structural Identification of Individual Helical Amyloid Filaments by Integration of Cryo-Electron Microscopy-Derived Maps in Comparative Morphometric Atomic Force Microscopy Image Analysis

The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297–391), termed ‘dGAE’. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are structurally closely related to the paired helical filaments (PHFs) isolated from Alzheimer’s disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.     

A novel canonical dual computational approach for prion AGAAAAGA amyloid fibril molecular modeling

Many experimental studies have shown that the prion AGAAAAGA palindrome hydrophobic region (113-120) has amyloid fibril forming properties and plays an important role in prion diseases. However, due to the unstable, noncrystalline and insoluble nature of the amyloid fibril, to date structural information on AGAAAAGA region (113-120) has been very limited. This region falls just within the N-terminal unstructured region PrP (1-123) of prion proteins. Traditional X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy experimental methods cannot be used to get its structural information. Under this background, this paper introduces a novel approach of the canonical dual theory to address the 3D atomic-resolution structure of prion AGAAAAGA amyloid fibrils. The novel and powerful canonical dual computational approach introduced in this paper is for the molecular modeling of prion AGAAAAGA amyloid fibrils, and that the optimal atomic-resolution structures of prion AGAAAAGA amyloid fibils presented in this paper are useful for the drive to find treatments for prion diseases in the field of medicinal chemistry. Overall, this paper presents an important method and provides useful information for treatments of prion diseases.     

On the Structural Diversity and Individuality of Polymorphic Amyloid Protein Assemblies

The prediction of highly ordered three-dimensional structures of amyloid protein fibrils from the amino acid sequences of their monomeric self-assembly precursors constitutes a challenging and unresolved aspect of the classical protein folding problem. Because of the polymorphic nature of amyloid assembly whereby polypeptide chains of identical amino acid sequences under identical conditions are capable of self-assembly into a spectrum of different fibril structures, the prediction of amyloid structures from an amino acid sequence requires a detailed and holistic understanding of its assembly free energy landscape. The full extent of the structure space accessible to the cross-β molecular architecture of amyloid must also be resolved. Here, we review the current understanding of the diversity and the individuality of amyloid structures, and how the polymorphic landscape of amyloid links to biology and disease phenotypes. We present a comprehensive review of structural models of amyloid fibrils derived by cryo-EM, ssNMR and AFM to date, and discuss the challenges ahead for resolving the structural basis and the biological consequences of polymorphic amyloid assemblies.     

Heterogeneous triangular structures of human islet amyloid polypeptide (amylin) with internal hydrophobic cavity and external wrapping morphology reveal the polymorphic nature of amyloid fibrils

The misfolding and self-assembly of human islet amyloid polypeptide (hIAPP or amylin) into amyloid fibrils is pathologically linked to type II diabetes. The polymorphic nature of both hIAPP oligomers and fibrils has been implicated for the molecular origin of hIAPP toxicity to islet β-cells, but little is known about the polymorphic structure and dynamics of these hIAPP oligomers/fibrils at the atomic level. Here, we model the polymorphism of full length hIAPP(1-37) oligomers based on experimental data from solid-state NMR, mass per length, and electron microscopy using all-atom molecular dynamics simulation with explicit solvent. As an alternative to steric zipper structures mostly presented in the 2-fold symmetrical fibrils, the most striking structural feature of our proposed hIAPP oligomers is the presence of 3-fold symmetry along the fibril growth axis, in which three β-sheet-layers wind around a hydrophobic core with different periodicities. These 3-fold triangular hIAPP structures dramatically differ in the details of the β-layer assembly and core-forming sequence at the cross section, but all display a high structural stability with favorable layer-to-layer interactions. The 3-fold hIAPP structures can also serve as templates to present triple-stranded helical fibrils via peptide elongation, with different widths from 8.7 to 9.9 nm, twists from 2.8° to 11.8°, and pitches from 14.5 to 61.1 nm, in reasonable agreement with available biophysical data. Because similar 3-fold Aβ oligomers are also observed by both NMR experiments and our previous simulations, the 3-fold structure could be a general conformation to a broad range of amyloid oligomers and fibrils. Most importantly, unlike the conventional stacking sandwich model, the proposed wrapping-cord structures can readily accommodate more than three β-layers via a two dimension conformation search by rotating and translating the β-layers to adopt different favorable packings, which can greatly enrich the polymorphism of amyloid oligomers and fibrils.     

Peptide conformation and supramolecular organization in amylin fibrils: constraints from solid-state NMR

The 37-residue amylin peptide, also known as islet amyloid polypeptide, forms fibrils that are the main peptide or protein component of amyloid that develops in the pancreas of type 2 diabetes patients. Amylin also readily forms amyloid fibrils in vitro that are highly polymorphic under typical experimental conditions. We describe a protocol for the preparation of synthetic amylin fibrils that exhibit a single predominant morphology, which we call a striated ribbon, in electron microscopy and atomic force microscopy images. Solid-state nuclear magnetic resonance (NMR) measurements on a series of isotopically labeled samples indicate a single molecular structure within the striated ribbons. We use scanning transmission electron microscopy and several types of one- and two-dimensional solid-state NMR techniques to obtain constraints on the peptide conformation and supramolecular structure in these amylin fibrils and to derive molecular structural models that are consistent with the experimental data. The basic structural unit in amylin striated ribbons, which we call the protofilament, contains four layers of parallel beta-sheets, formed by two symmetric layers of amylin molecules. The molecular structure of amylin protofilaments in striated ribbons closely resembles the protofilament in amyloid fibrils with a similar morphology formed by the 40-residue beta-amyloid peptide that is associated with Alzheimer's disease.     

Solid-state NMR as a probe of amyloid structure

Solid state nuclear magnetic resonance (NMR) has developed into one of the most informative and direct experimental approaches to the characterization of the molecular structures of amyloid fibrils, including those associated with Alzheimer's disease. In this article, essential aspects of solid state NMR methods are described briefly and results obtained to date regarding the supramolecular organization of amyloid fibrils and the conformations of peptides within amyloid fibrils are reviewed.     

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.     

A molecular dynamics study of the interaction of D-peptide amyloid inhibitors with their target sequence reveals a potential inhibitory pharmacophore conformation

The self-assembly of soluble proteins and peptides into β-sheet-rich oligomeric structures and insoluble fibrils is a hallmark of a large number of human diseases known as amyloid diseases. Drugs that are able to interfere with these processes may be able to prevent and/or cure these diseases. Experimental difficulties in the characterization of the intermediates involved in the amyloid formation process have seriously hampered the application of rational drug design approaches to the inhibition of amyloid formation and growth. Recently, short model peptide systems have proved useful in understanding the relationship between amino acid sequence and amyloid formation using both experimental and theoretical approaches. Moreover, short d-peptide sequences have been shown to specifically interfere with those short amyloid stretches in proteins, blocking oligomer formation or disassembling mature fibrils. With the aim of rationalizing which interactions drive the binding of inhibitors to nascent β-sheet oligomers, in this study, we have carried out extensive molecular dynamics simulations of the interaction of selected d-peptide sequences with oligomers of the target model sequence STVIIE. Structural analysis of the simulations helped to identify the molecular determinants of an inhibitory core whose conformational and physicochemical properties are actually shared by nonpeptidic small-molecule inhibitors of amyloidogenesis. Selection of one of these small molecules and experimental validation against our model system proved that it was indeed an effective inhibitor of fibril formation by the STVIIE sequence, supporting theoretical predictions. We propose that the inhibitory determinants derived from this work be used as structural templates in the development of pharmacophore models for the identification of novel nonpeptidic inhibitors of aggregation.     

Structural analysis of oligomeric and protofibrillar Aβ amyloid pair structures considering F20L mutation effects using molecular dynamics simulations

Aβ amyloid proteins are involved in neuro‐degenerative diseases such as Alzheimer's, Parkinson's, and so forth. Because of its structurally stable feature under physiological conditions, Aβ amyloid protein disrupts the normal cell function. Because of these concerns, understanding the structural feature of Aβ amyloid protein in detail is crucial. There have been some efforts on lowering the structural stabilities of Aβ amyloid fibrils by decreasing the aromatic residues characteristic and hydrophobic effect. Yet, there is a lack of understanding of Aβ amyloid pair structures considering those effects. In this study, we provide the structural characteristics of wildtype (WT) and phenylalanine residue mutation to leucine (F20L) Aβ amyloid pair structures using molecular dynamics simulation in detail. We also considered the polymorphic feature of F20L and WT Aβ pair amyloids based on the facing β‐strand directions between the amyloid pairs. As a result, we were able to observe the varying effects of mutation, polymorphism, and protofibril lengths on the structural stability of pair amyloids. Furthermore, we have also found that opposite structural stability exists on a certain polymorphic Aβ pair amyloids depending on its oligomeric or protofibrillar state, which can be helpful for understanding the amyloid growth mechanism via repetitive fragmentation and elongation mechanism. Proteins 2017; 85:580–592. © 2016 Wiley Periodicals, Inc.     

Instantaneous amyloid fibril formation of alpha-synuclein from the oligomeric granular structures in the presence of hexane

Amyloid fibrils found in various neurodegenerative disorders are also recognized as high-performance protein nanomaterials with a formidable rigidity. Elucidation of an underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop controlling strategy toward the diseases, but also to apply the protein fibrils for future nanobiotechnology. alpha-Synuclein is an amyloidogenic protein responsible for the radiating filament formation within Lewy bodies of Parkinson's disease. The amyloid fibril formation of alpha-synuclein has been shown to be induced from the oligomeric granular species of the protein acting as a growing unit by experiencing structural rearrangement within the preformed oligomeric structures in the presence of an organic solvent of hexane. This granule-based concerted amyloid fibril formation model would parallel the prevalent notion of nucleation-dependent fibrillation mechanism in the area of amyloidosis.     

Development of the Structural Core and of Conformational Heterogeneity during the Conversion of Oligomers of the Mouse Prion Protein to Worm-like Amyloid Fibrils

Understanding how structure develops during the course of amyloid fibril formation by the prion protein is important for understanding prion diseases. Determining how conformational heterogeneity manifests itself in the fibrillar and pre-fibrillar amyloid aggregates is critical for understanding prion strain phenotypes. In this study, the formation of worm-like amyloid fibrils by the mouse prion protein has been characterized structurally by hydrogen-deuterium exchange coupled to mass spectrometry. The structural cores of these fibrils and of the oligomer on the direct pathway of amyloid fibril formation have been defined, showing how structure develops during fibril formation. The structural core of the oligomer not on the direct pathway has also been defined, allowing the delineation of the structural features that make this off-pathway oligomer incompetent to directly form fibrils. Sequence segments that exhibit multiple local conformations in the three amyloid aggregates have been identified, and the development of structural heterogeneity during fibril formation has been characterized. It is shown that conformational heterogeneity is not restricted to only the C-terminal domain region, which forms the structural core of the aggregates; it manifests itself in the N-terminal domain of the protein as well. Importantly, all three amyloid aggregates are shown to be capable of disrupting lipid membrane structure, pointing to a mechanism by which they may be toxic.     

Instantaneous Amyloid Fibril Formation of α-Synuclein from the Oligomeric Granular Structures in the Presence of Hexane

Amyloid fibrils found in various neurodegenerative disorders are also recognized as high-performance protein nanomaterials with a formidable rigidity. Elucidation of an underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop controlling strategy toward the diseases, but also to apply the protein fibrils for future nanobiotechnology. α-Synuclein is an amyloidogenic protein responsible for the radiating filament formation within Lewy bodies of Parkinson's disease. The amyloid fibril formation of α-synuclein has been shown to be induced from the oligomeric granular species of the protein acting as a growing unit by experiencing structural rearrangement within the preformed oligomeric structures in the presence of an organic solvent of hexane. This granule-based concerted amyloid fibril formation model would parallel the prevalent notion of nucleation-dependent fibrillation mechanism in the area of amyloidosis.     

Molecular modeling of the core of Abeta amyloid fibrils

Amyloid fibrils, a key pathological feature of Alzheimer's disease (AD) and other amyloidosis implicated in neurodegeneration, have a characteristic cross-beta structure. Here we present a structural model for the core of amyloid fibrils formed by the Abeta peptide using computational approaches and experimental data. Abeta(15-36) was threaded against the parallel beta-helical proteins. Our multi-layer model was constructed using the top scoring template 1lxa, a left-handed parallel beta-helical protein. This six-rung helical model has in-register repeats of the Abeta(15-36) sequence. Each rung has three beta-strands separated by two turns. The model was tested using molecular dynamics simulations in explicit water, and is in good agreement with a number of experimental observations. In addition, a model based on right-handed helical proteins is also described. The core structural model described here might serve as the building block of the Abeta(1-40) amyloid fibril as well as some other amyloid fibrils.     

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.     

Kinetic intermediates of amyloid fibrillation studied by hydrogen exchange methods with nuclear magnetic resonance

Amyloid fibrils with an ordered cross-β structure are one form of protein aberrant aggregates. Fibrils themselves and on-pathway small aggregates are involved in many neurodegenerative diseases and amylodoses. Over the past decade, much has been learned about the conformation of amyloid fibrils by using various biochemical and biophysical approaches. Amyloid fibrils accommodate rigid core structures composed of regular intra- and intermolecular non-covalent bonds such as hydrogen bonds, and disordered flexible regions exposed to solvents. In contrast to the improved understanding of fibril structures, few studies have investigated the short-living monomeric intermediates which interact with amyloid fibrils for elongation and the self-associated intermediates in the course of amyloidogenesis at the residue level. To study static fibrillar structures and kinetic intermediates, hydrogen/deuterium exchange (HD(ex)) coupled with solution-state NMR spectroscopy is one of the most powerful methods with a high time and atomic resolution. Here, we review studies on the structural properties of amyloid fibrils based on a combination of dimethylsulfoxide-quenched HD(ex) and NMR spectroscopy. Recent studies on transient kinetic intermediates during fibril growth by means of pulse-labeling HD(ex) aided by a quenched-flow apparatus and NMR spectroscopy are focused on.